Bioengineered human corneal stromal tissue

ABSTRACT

Provided herein is a method of making an aligned ECM scaffold useful in refractive correction of the eye and repair of the cornea. Methods of use of the scaffold as well as a scaffold construct are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/581,707, filed Nov. 9, 2012, which is a National Stage ofInternational Patent Application No. PCT/US2011/027195, filed Mar. 4,2011, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/310,559, filed Mar. 4, 2010, each ofwhich is incorporated herein by reference in its entirety.

This invention was made with government support under Grant No. EY016415awarded by the National Institutes of Health. The government has certainrights in the invention.

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and is hereby incorporated by referenceinto the specification in its entirety. The name of the text filecontaining the Sequence Listing is 6527_1701538_ST25.txt. The size ofthe text file is 1,654 Bytes, and the text file was created on Mar. 1,2017.

Provided herein are compositions and devices, and in particular,biodegradable scaffolds useful in the preparation of bioengineeredcorneal tissue. Also provided herein are methods of making and using thescaffolds.

The appropriate functionality of any organ and tissue in human bodyrelies on the complex structural organization of cells and extracellularmatrix (ECM). ECM, featuring the special three-dimensional (3D)structures, renders organs and tissues mechanical functions. Moreimportantly, ECM provides the micro-environment to guide the cellularactivities, including cell spreading, migration, proliferation anddifferentiation, mainly due to that cells are sensitive to surfacetopology, molecular composition, and mechanical properties of thematrix. To mimic native cellular microenvironments is pivotal to controlthe cell-matrix interaction and recapitulate tissue architecture intissue engineering. Collagen is the most abundant protein found inanimal connective tissue. It is the main structural componentscomprising ECM to maintain the shape and integrity of tissues, andimpart mechanical strength. Although in part tissue-specific, collagensare preferably aligned in constitute human tissues, including compactbone, tendons and ligaments, articular cartilage, among others. In thecorneal stroma, hybrid type-I/V collagen fibrils form a spatiallyorganized pseudo hexagonal lattice, in which the uniform diameter andspacing of collagen fibrils are 30.8 nm and 55.3 nm, respectively (Meek,K. M., et al. Biophys. J. 1993, 64, 273-280 and Meek, K. M., et al. Exp.Eye Res. 2004, 78, 503-512). The alignment of collagen fibers ofconsecutive lamellae is perpendicular to each other, resulting in atransparent lens capable of resisting external trauma and supportingintraocular pressure. Once the corneal stroma is injured, quiescentkeratocytes residing within the stroma are activated to differentiateinto myofibroblasts, which secrete altered disorganized collagenousmatrix, resulting in stromal scar formation and reduced transparency.Clearly, to constitute the aligned collagen-based ECM with nano-scalespatial organization is critical to succeed in repair and regenerationof the damaged and diseased corneal stroma tissue.

Although it is very challenging to create three-dimensional (3D) orderlycollagen-fibril construct, its importance in tissue engineering hasattracted more and more attention of biomaterial scientists to developmethods to align collagen in vitro. Contact guidance is the simple andeffective approach to provide the physical cue to direct cellorientation and organization of cell-secreted collagens. Recently,Guillemette et al. found micro-patterned surfaces can guide the cells toalign along the grooves, leading to cell-secreted collagens to organizealong cell orientation (Integr Biol (Camb) 2009, 1, 196-204).Interestingly, the subsequent organization of cells and cell-secretedcollagens is cell-type specific following the alignment of the firstlayer of cells and cell-secreted collagen. In contrast to dermalfibroblasts, corneal fibroblast do not lose the alignment from thesecond layer. However, transmission electron micrographs (TEM) revealthat the collagen fibrils in the construct is diameter-polydispersed,and lack long-range order. Clearly, not only the tissue origin of thecells, but also the cell phenotype plays the crucial role in thenanoscale spatial organization of cell-secreted collagen fibrils.

Keratocytes are the inborn cell population in human corneal stroma,responsible for secreting a spectrum of unique matrix molecules, e.g.keratocan and keratan sulfate, that constitute the transparent stromatissue, a well-organized collagen-based 3-dimensional nano-construct.When attempts are made to expand keratocyte populations in culture inserum-based medium, the keratocytes inevitably lose their phenotype anddifferentiate into fibroblasts, leading to the formation of scar tissue.Accordingly, there is a need of a population of progenitor cells whichcan be expanded in culture, and then differentiated into keratocytesthat retain the ability to produce an appropriate extracellular matrix.Thus, there is a need for biodegradable materials that combine thefavorable bioreactive and biocompatible properties ofnaturally-occurring scaffold materials with the reproducible andpredictable properties of synthetic scaffold materials. There is also aneed for biocompatible and biodegradable materials that are useful forpromoting wound and tissue healing that possess bioactive components,and that exhibit mechanical properties similar to native tissue.

SUMMARY

Provided herein are template scaffolds that are useful in preparing asuitable bioscaffold for implantation in the human cornea. The scaffoldmaterials produced by the methods described herein and using thematerials described herein can be implanted in a patient's cornea eitherto correct refractive defects, such as presbyopia, hyperopia, myopia andastigmatism, or as a replacement of scarred or otherwise damaged ordefective stromal tissue.

In one embodiment, a method is provided for producing a bioscaffold forimplantation in the cornea of a patient, for instance as a corneal inlayor onlay. The method comprises culturing functional keratocytes on ascaffold template comprising one or more layers comprising aligned (thatis a predominance of fibers in the layer are substantially parallel toeach other, such as are formed by electrospinning techniques asdescribed herein) fibers of a biocompatible, biodegradable polymericcomposition that is optionally elastomeric, where when more than onelayer is present in a plurality of layers, the fibers of two or morelayers, such as adjacent layers, are aligned at different angles, and inone embodiment at 20° to 90°, including increments therebetween, and inone embodiment, at 45° or perpendicular (orthogonally) to each other.The functional keratocytes are cultured on the scaffold for a length oftime sufficient for the cells to produce an aligned and preferablytransparent product ECM scaffold, which substantially replaces thebiocompatible, biodegradable polymeric composition. The ECM scaffold isthen optionally processed into a defined shape, such as a disc forcorneal inlay or onlay, if the original scaffold is not an appropriatesize or shape for its intended end-use. In one embodiment, the constructthus produced is implanted into the eye of a patient. In one embodiment,the produced construct is decellularized prior to implantation. Whenimplanted in the eye of a patient, the decellularized scaffold ispopulated with native cells. The implantation can be an onlay, an inlayor can replace native stromal tissue either in part or wholly.

In one embodiment of the described method, the functional keratocytesare produced by differentiation of progenitor cells, e.g., stem cells(multipotent cells), capable of differentiating into the functionalkeratocytes by culturing in a keratocyte differentiation medium. Thestem cells can be any stem cell able to differentiate into functionalkeratocytes that produce collagen, keratan sulfate and keratocan.Examples of suitable stem cells are corneal stromal stem cells andadipose-derived stem cells, and in one embodiment, human corneal stromalstem cells and human adipose-derived stem cells. In one preferredembodiment, the stem cells are human corneal stromal stem cells.

In another embodiment, a bioscaffold template is provided comprising oneor more layers comprising aligned fibers of a biocompatible,biodegradable polymeric composition that is optionally elastomeric,where when more than one layer is present in a plurality of layers, thefibers of two or more layers, such as adjacent layers, are aligned atdifferent angles, and in one embodiment at 20° to 90°, includingincrements therebetween, and in one embodiment, at 45° or perpendicular(orthogonally) to each other. Adipose or corneal stem cells aredispersed within the template scaffold. In yet another embodiment, abioreactor is provided comprising the template scaffold in a culturevessel comprising keratocyte differentiation medium.

Material that is useful in preparing the scaffolds and cell constructsdescribed herein are biodegradable and biocompatible, includingpolyesters and polyurethanes comprising hydrophilic groups, such asether and ester groups.

As used herein, the terms “comprising,” “comprise” or “comprised,” andvariations thereof, in reference to elements of an item, composition,apparatus, method, process, system, etc. are meant to be open-ended,indicating that the item, composition, apparatus, method, process,system, etc. includes those elements and that other elements can beincluded and still fall within the scope/definition of the describeditem, composition, apparatus, method, process, system, etc.

The scaffold may comprise a therapeutic agent. For example and withoutlimitation, the therapeutic agent may be an antimicrobial agent chosenfrom one or more of: isoniazid, ethambutol, pyrazinamide, streptomycin,clofazimine, rifabutin, fluoroquinolones, ofloxacin, sparfloxacin,rifampin, azithromycin, clarithromycin, dapsone, tetracycline,erythromycin, ciprofloxacin, doxycycline, ampicillin, amphotericin B,ketoconazole, fluconazole, pyrimethamine, sulfadiazine, clindamycin,lincomycin, pentamidine, atovaquone, paromomycin, diclazaril, acyclovir,trifluorouridine, foscarnet, penicillin, gentamicin, ganciclovir,iatroconazole, miconazole, Zn-pyrithione, and silver salts such aschloride, bromide, iodide and periodate. Optionally, the therapeuticagent may be a growth factor, for example and without limitation, agrowth factor chosen from one or more of: basic fibroblast growth factor(bFGF), acidic fibroblast growth factor (aFGF), vascular endothelialgrowth factor (VEGF), hepatocyte growth factor (HGF), insulin-likegrowth factors 1 and 2 (IGF-1 and IGF-2), platelet derived growth factor(PDGF), stromal derived factor 1 alpha (SDF-1 alpha), nerve growthfactor (NGF), ciliary neurotrophic factor (CNTF), neurotrophin-3,neurotrophin-4, neurotrophin-5, pleiotrophin protein (neuritegrowth-promoting factor 1), midkine protein (neurite growth-promotingfactor 2), brain-derived neurotrophic factor (BDNF), tumor angiogenesisfactor (TAF), corticotrophin releasing factor (CRF), transforming growthfactors α and β (TGF-α and TGF-β), interleukin-8 (IL-8),granulocyte-macrophage colony stimulating factor (GM-CSF), interleukins,and interferons. The thereapeutic agent may be cellular, for example andwithout limitation one or more of stem cells, precursor stem cells,smooth muscle cells, skeletal myoblasts, myocardial cells, endothelialcells, and genetically modified cells.

The template scaffold can be prepared by any useful method, such as,without limitation, by casting or electrospinning, includingcombinations thereof. In a useful electrospinning method, the syntheticpolymeric component and a biological polymeric component can besuspended independently or together in a solvent and may therefore bespun together or independently (using, for example two nozzles) to forman scaffold.

Methods of promoting wound healing or tissue generation or regenerationin a patient also are provided. The methods comprise, withoutlimitation, implanting an scaffold as described herein at or near a sitefor wound healing or tissue generation or regeneration in the patient.Likewise a method of promoting wound healing or tissue generation orregeneration in a patient is provided comprising contacting an scaffoldas described herein with cells in vitro (for instance, ex vivo forautologous cells), culturing the cells in vitro so that the cells growin and/or on the scaffold; and implanting the scaffold at or near a sitefor wound healing or tissue generation or regeneration in the patient.In either method, the scaffold may comprise a therapeutic agent asdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic flow diagram illustrating a method of makingan ECM scaffold for cornea implantation.

FIGS. 2A-2F. Morphologies of hCSSCs seeded on two kinds of PEUUscaffolds. The surface morphologies of the two scaffolds werecharacterized by scanning electron microscope (SEM): FIG. 2A (a) alignednanofibrous PEUU sheet, whose fibers are 165±55 nm in diameter; FIG. 2D(d) PEUU cast film. Cellular viability and morphology was evaluatedemploying Calcein AM, and imaged by confocal laser-scanning microscope(CLSM). FIG. 2B (b) and FIG. 2C (c) are the fluorescent micrographs ofhCSSCs seeded on aligned nanofibrous PEUU sheet for 1-day and 3-day cellseeding, respectively. FIG. 2D (d) and FIG. 2E (e) are those seeded onPEUU cast film for 1-day and 3-day, respectively.

FIG. 3. Changes in gene expression of hCSSCs seeded on scaffolds: (

) cast film and) (

) aligned nano-fibrous sheet. mRNA abundance was compared with hCSSCscultured in SCGM (----). Ratios of abundance of each transcript betweenhCSSCs seeded on different scaffolds cultured in KDM and in SCGM areexpressed on a log scale. Since KERA has no expression in hCSSCscultured in SCGM, it is expressed in linear plot.

FIGS. 4A-4B. Two-photon images of hCSSCs-secreted extracelluar matrix(ECM) varying with scaffolds: FIG. 4A (a) aligned nano-fibrous PEUUscaffold, and FIG. 4B (b) cast PEUU film. The second harmonic generationsignal for collagens when excited at λ=840 nm is red. Nuclei are stainedgreen.

FIGS. 5A-5D. SEM micrographs of hCSSCs and hCSSCs-secreted ECM on thevarying scaffolds. The morphologies of hCSSCs and hCSSCs-secreted ECMwere detailed from micron-scale to nano-scale with increasingmagnification: FIGS. 5A and 5B (a,b) for aligned nano-fibous PEUU sheet,and FIGS. 5C and 5D (c,d) for cast PEUU film with increasingmagnification. The banded structures along the fibrils are disclosed ininserted parts of FIG. 5B (b) and FIG. 5D (d).

FIGS. 6A-6H. Transmission electron micrographs of hCSSCs seeded onaligned nano-fibrous PEUU sheets (FIGS. 6A-6C, (a-c)) and cast PEUUfilms (FIGS. 6G-6H, (g-h)) for six weeks. TEM micrographs (FIG. 6A (a)and FIG. 6B (b)) revealed fibers from cells on the aligned scaffoldexhibited high fidelity to a preferred orientation and long-range order.The fibers were normal to the viewing plane when the section crossed thecell long axis (FIGS. 6A and 6B), whereas when the section was along thecell long axis, fibers were nearly parallel to each other. Digitalanalysis of the fiber diameter, fiber spacing and fiber orientation areseen in FIGS. 6D (d), 6E (e), and 6F (f), respectively. Fibers generatedon the cast film randomly distributed in the construct (FIGS. 6G and6H). The scale bar in the insert is 100 μm.

FIGS. 7A-7E and FIGS. 7A′-7E′. Immunofluorescence micrographs ofhCSSC-secreted ECM on (FIGS. 7A-7E, (a-e)) aligned nano-fibrous PEUUsheet and (FIGS. 7A′-7E′, (a′-e′)) cast PEUU film after six weeksculture: (FIGS. 7A, 7A′, (a, a′)) collagen I, (FIGS. 7B, 7B′, (b, b′))collagen V, (FIGS. 7C, 7C′, (c, c′)) collagen VI, (FIGS. 7D, 7D′, (d,d′)) keratan sulfate and (FIGS. 7E, 7E′, (e, e′)) keratocan. Nuclei werestained with DAPI and appear blue.

FIGS. 8 and 9 are SEM micrographs of aligned nano-fibril sheets preparedaccording to the conditions indicated at 1000× and 30,000×,respectively.

FIGS. 10A and 10B show, respectively a top and an elevated view of acell culture chamber described in Example 2.

FIGS. 11 and 12 are TEM micrographs (axial cross section, FIG. 11 andcross section across the axis, FIG. 12) of cells grown according toExample 2.

FIGS. 13 and 14A-14G are SEM and TEM micrographs respectively of cells 6weeks after differentiation. as described in Example 2.

FIGS. 15A and 15B. Flow cytometric identification of side populationfrom cultured human adipose-derived stem cells (ADSC). FIG. 15A:Passage-two ADSC stained with Hoechst 33342 were analyzed using 350-nmexcitation with blue (635 nm) and red (488 nm) emission. Cells showingreduction of both blue and red fluorescence (side population cells) wereanalyzed as defined by the box outlined on the left. FIG. 15B: Ananalysis similar to FIG. 15A but with a preincubation in 50 μM verapamilbefore incubation with Hoechst 33342.

FIGS. 16A-16E. Induction of adipocytes from ADSC. ADSC were induced todifferentiate into adipocytes in ADM for two weeks as described inMethods. FIG. 16A, FIG. 16B: ADSC were fixed and stained with Oil Red O.FIG. 16C, FIG. 16D: ADSC without induction were stained with Oil Red Oas control. FIG. 16E: mRNA for leptin was detected by RT-PCR. Leptinexpression (Upper) and 18S (Lower). Lane 1, uncultured keratocytes; lane2, ADSC; lane 3, ADSC in adipocyte induction medium. Scale bars: 100 μm(FIG. 16A, FIG. 16C); 50 μm (FIG. 16B, FIG. 16D).

FIGS. 17A and 17B. Induction of cartilage matrix expression by ADSC.ADSC (FIG. 17A) and CF (FIG. 17B) were cultured as pellets (2×105) inchondrocyte differentiation medium for three weeks. The pellets werefixed, imbedded in OCT, cut into 8 μm thick sections and stained withtoluidine blue to detect proteoglycan staining typical of cartilage.Scale bar indicates 50 μm.

FIGS. 18A-18E. Induction of keratocyte markers in ADSC. FIG. 18A, FIG.18B: ADSC were cultured in fibrin gels for 3 weeks in keratocytedifferentiation medium. FIG. 18C, FIG. 18D: ADSC were cultured as pelletfor 3 weeks in keratocyte differentiation medium Immunofluorescentstaining shows the presence of keratan sulfate with antibody J19 (green;FIG. 18A, FIG. 18C) or keratocan with antibody KeraC, (green; FIG. 18B,FIG. 18D). Red shows nuclear staining by propidium iodide. FIG. 18E:RT-PCR shows keratocan expression in (left to right) unculturedkeratocytes (positive control), ADSC in fibrin gel, ADSC as pelletculture, ADSC in SCGM. Scale bars=20 μm.

FIGS. 19A-19D. Keratan sulfate, keratocan protein, and mRNA expressionby CSSC, ADSC, and CF in different media. FIG. 19A, FIG. 19B: Fibrin gelcultures after 3 weeks. FIG. 19C, FIG. 19D: Pellet cultures after 3weeks. FIG. 19A and FIG. 19C are western blots showing keratan sulfateand keratocan. FIG. 19B and FIG. 19D show qPCR data of keratocan mRNA.Samples 1-3: CF, 4-6: CSSC cells; 7-9, ADSC cells. Samples 1, 4, 7:keratocyte differentiation medium; Samples 2, 5, 8: keratocytedifferentiation medium+1% HSHS. Samples 3, 6, 9: DMEM/F-12 medium withbovine corneal extract (1:10). Expression of mRNA is shown normalized tomonolayer of CSSC in keratocyte differentiation medium=100.

FIG. 20. ALDH is upregulated in ADSC cultured under conditions thatinduce keratocyte differentiation. Expression of ALDH3A1 was compared inADSC cells cultured as described in FIGS. 19A-19D using qPCR asdescribed in Methods. Expression levels were normalized to that of ADSCin SCGM (ADSC-Ctrl) in samples in Fibrin or Pellets under conditions 7,8, 9 as described in FIGS. 19A-19D.

FIG. 21. Keratan sulfate staining in CSSC, ADSC, and CF cultured infibrin gels or as pellets in different media. Frozen sections of pelletand fibrin gel cultures were stained with antibody J19 against keratansulfate (green) and nuclei (red) after 3 weeks of culture in differentmedia. Abbreviations: CSSC: corneal stromal stem cells, ADSC:Adipose-derived stem cell, Adv: Advanced DMEM, FGF: fibroblast growthfactor 2, HS: heparin stripped horse serum, CE: bovine corneal extract.Scale bars=50 μm.

FIG. 22. Keratocan staining in CSSC, ADSC, and CFs cultured in differentmedia. Frozen sections of pellet and fibrin gel cultures were stainedwith antibody KeraC for keratocan (green) and cell nuclei (red) after 3weeks of culture in several different media. Abbreviations: CSSC:corneal stromal stem cells, ADSC: Adipose-derived stem cell, Adv:Advanced DMEM, FGF: fibroblast growth factor 2, HS: heparin strippedhorse serum, CE: bovine corneal extract. Scale bar=50 μm.

FIGS. 23A and 23B. Changes in gene expression of ADSCs (week 2, 4, 6)seeded on scaffolds. ADSCs on trans-well used as the control since theywere incubated with stem cell growth medium (SCGM). 18S and GAPDH wereused as qualitative external controls.

FIG. 24. Immunofluorescence microscopy images of ADSCs-secreted ECM onaligned nano-fibrous PEUU sheet.

FIG. 25. Two-photon images of ADSCs secreted ECM on aligned nano-fibrousPEUU scaffold. The red was the second harmonic generation signal forcollagens when excited at wavelength, λ=840 nm, but without staining.The green in week 2 and 4 was the nuclei stained with Sytox Green. Thegreen in week 6 was the whole cell stained with CellTracker Green.

FIG. 26. SEM micrographs of ADSCs and ADSCs-secreted ECM on thescaffolds. The morphologies of ADSCs and ADSCs-secreted ECM weredetailed from micron-scale to nano-scale with increasing magnificationas shown above.

FIG. 27. Transmission electron micrographs of hCSSCs PEUU scaffoldsseeded on aligned nano-fibrous PEUU sheet. When samples were microtomedalong the fiber long axes, few fibrils were parallel to each other;however, mostly were fragmented.

FIG. 28. Western blot of collected media for 6 weeks. The membrane wassubjected to immunoblotting with J19 antibody against keratan sulfate.

DETAILED DESCRIPTION

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of these ranges isintended as a continuous range including every value between the minimumand maximum values. For definitions provided herein, those definitionsrefer to word forms, cognates and grammatical variants of those words orphrases. “A,” “an,” and “one” include the plural unless indicatedotherwise.

The copolymers, compositions and components thereof are preferablybiocompatible. By “biocompatible,” it is meant that a polymercomposition and its normal in vivo degradation products arecytocompatible and are substantially non-toxic and non-carcinogenic in apatient within useful, practical and/or acceptable tolerances. By“cytocompatible,” it is meant that the copolymers or compositions aresubstantially non-toxic to cells and typically and most desirably cansustain a population of cells and/or the polymer compositions, devices,copolymers, and degradation products thereof are not cytotoxic and/orcarcinogenic within useful, practical and/or acceptable tolerances. Forexample, a copolymer composition when placed in a human cornea stem cellculture does not adversely affect the viability, growth, adhesion, andnumber of cells. In one non-limiting example, the co-polymers,compositions, and/or devices are “biocompatible” to the extent they areacceptable for use in a human veterinary patient according to applicableregulatory standards in a given legal jurisdiction. In another examplethe biocompatible polymer, when implanted in a patient, does not cause asubstantial adverse reaction or substantial harm to cells and tissues inthe body, for instance, the polymer composition or device does not causenecrosis or an infection resulting in harm to tissues organs or theorganism from the implanted compositions.

As used herein, a “polymer” is a compound formed by the covalent joiningof smaller molecules, which are referred to herein as residues, orpolymer subunits, when incorporated into a polymer. A “copolymer” is apolymer comprising two or more different residues. Prior toincorporation into a polymer, the residues typically are described asmonomers. Non-limiting examples of monomers, in the context ofacrylic/polyester copolymers described herein, include: acrylic oracrylamide monomers, such as acrylic acid, isopropylacrylamide,acrylamide, acrylic N-hydroxysuccinimide ester and hydroxyethylmethacrylate, lactide, and trimethylene carbonate. A monomer may be amacromer prepared from even smaller monomers, such as a hydroxyethylmethacrylate-polylactide (HEMAPLA) macromer or hydroxyethylmethacrylate-poly(trimethylene carbonate) (HEMAPTMC) macromer.

The biological scaffolds described herein can be used for a large numberof medical applications including, but not limited to refractive surgeryto alter the refractivity of the cornea and to repair or replace cornealstroma.

FIG. 1 shows a general outline of a method described herein. In themethod, stem cells are seeded onto a scaffold in which polymer fibersare aligned and preferably allowed to adhere to and optionally propagateon the single sheet. The seeded sheets are then stacked at differentangles, meaning the orientation of the fibers of adjacent sheets aredifferent (that is, non-parallel) to each-other. Though FIG. 1 shows anorthogonal arrangement, the sheets can be oriented at any angle withrespect to each other that is not 0°. As would be recognized by one ofordinary skill a perfectly perpendicular arrangement or arrangement atany specified angle is not necessarily achievable or practicable, nor isperfect alignment of the fibers within a sheet. Therefore, theorthogonal or perpendicular (90°) arrangement, or any angle, includessome acceptable deviation from stated angle and include orientationsthat are essentially at that angle or substantially at that angle (θ),such as θ±20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2° or 1° so long asthe scaffold retains its ability to form acceptable product cellularconstructs or ECM scaffolds.

At some point, either before the sheets are stacked, or afterwards, thestem cells are differentiated by adding suitable differentiation mediumto the culture. As indicated below, for the purpose of producing aproduct corneal stroma ECM structure, the stem cells are hCSSCs andkeratocyte differentiation medium (KDM) is added. Exemplary KDM isdescribed below in the Examples, and is suitable for differentiatinghCSSCs to corneal keratocytes, and includes, for example an ascorbate,such as ascorbic acid or ascorbic acid phosphate (e.g. A2P, below) andoptionally TGFβ3, insulin and/or basic fibroblast growth factor.Ascorbate is recognized as being able to elicit keratocyte-like matrixproduction in vitro. It may be desirable also to add TGF-β3, or othergrowth factors to the medium to culture hCSSCs, ADSCs or other stem celltypes.

Once the layers are stacked, the resultant multi-layered scaffold ismaintained in culture for a length of time sufficient for the polymerictemplate scaffold to dissolve or substantially dissolve and for theproduct ECM scaffold to form.

In an alternate embodiment, the sheets are stacked after the product ECMis produced in a single-layer sheet, either with the cells or afterdecellularization. Controlled compression can be used to laminate sheetstogether.

According to one non-limiting embodiment, for each layer of fibers, thefiber diameter ranges from 100 to 200 nm in thickness, with spacingbetween fibers of less than 1 μm. Each single layers typically rangefrom 5 μm-10 μm in thickness, for example 8 μm on the average. Normalhuman cornea stroma is about 500 μm in thickness, meaning approximately50-60 layers would be needed to produce a full-thickness scaffold.Controlled compression can be used to fuse together monolayers oriented,for example, orthogonally on a layer-by-layer basis, or the templatescaffold and cells can be alternately electrodeposited andelectrosprayed until a desired thickness is achieved. In this case, thepolymer composition and cells are not electrospun on a mandrel but canbe electrodeposited on a planar surface with alternate groundingelectrodes arranged so that alternate layers of fibers are deposited ata desired angle with respect to each other, for instance and withoutlimitation, 90° or 45°.

In one variation of this method, hCSSCs or other stem cells aredifferentiated prior to seeding into functional keratocytes and thefunctional keratocytes are seeded onto the template scaffold prior tostacking. The cells are cultured in a KDM.

Once the product ECM scaffold is formed, it may be further processed forimplantation. Due to the immune privileged nature of the eye, allogeneicor even xenogeneic cells may be transferred in the product scaffold andcan survive within the eye. That said, it may be most desirable todecellularized and sterilize the scaffold prior to implanting thescaffold, as outlined below. The product ECM scaffold is processed intoa structure that is suitable for implantation. A large variety ofmethods may be used to process the shape and thickness of the productECM scaffold. Circular section may be punched or cut from the material.Multiple layers of varying sized can be stacked and annealed orlaminated to provide a thicker construct.

A variety of cells can be used in the methods described herein. In oneexample, hCSSCs are seeded onto the structure and then aredifferentiated to produce functional keratocytes. Alternately, thehCSSCs are pre-differentiated in a KDM and then are seeded onto thestructure. As indicated below, the readily-available ADSCs are promisingcandidates for seeding and later differentiation into functionalkeratocytes. Because the cells are used to form the ECM, but are notnecessarily required to be implanted in the eye, xenogeneic cornealstroma stem cells may be used, and may be preferable to hCSSCs, givenproduct ECM scaffolding produced by CSSCs of other species may prove tobe equally or more suitable for the purposes described herein thanhuman. Likewise, stem cells from other human organs or tissue, or fromother species may prove to be acceptable in the uses described herein,as is the case with ADSCs. hCSSCs may be prepared according to themethods described below. Non-human hCSSCs have been cultured and, forexample, can be isolated and propagated according to the same or similarmethods. Adipose-derived stem cells ADSCs, can be prepared by any usefulmethod, including that shown below. U.S. Pat. Nos. 6,777,231 and7,470,537, each of which is incorporated herein by reference in itsentirety, describe adipose-derived stem cells and methods of makingadipose-derived stem cells.

As used herein, a progenitor cell is a cell type in a cell lineage thatcan differentiate into another cell type in that lineage. Cornealstromal stem cells isolated substantially according to the methodsdescribed herein can be identified by expression of ABCG2 and have theability to differentiate into functional keratocytes in keratocytedifferentiation media as described in the examples below.

As used herein, the term “polymer” refers to both synthetic polymericcomponents and biological polymeric components. “Biological polymer(s)”are polymers that can be obtained from biological sources, such as,without limitation, mammalian or vertebrate tissue, as in the case ofcertain extracellular matrix-derived (ECM-derived) compositions.Biological polymers can be modified by additional processing steps.Polymer(s), in general include, for example and without limitation,mono-polymer(s), copolymer(s), polymeric blend(s), block polymer(s),block copolymer(s), cross-linked polymer(s), non-cross-linkedpolymer(s), linear-, branched-, comb-, star-, and/or dendrite-shapedpolymer(s), where polymer(s) can be formed into any useful form, forexample and without limitation, a hydrogel, a porous mesh, a fiber,woven mesh, or non-woven mesh, such as, for example and withoutlimitation, as a non-woven mesh formed by electrospinning.

Generally, the polymeric components suitable for preparation of thetemplate scaffold described herein may be any polymer that isbiocompatible and is either biodegradable or has a Lower CriticalSolution Temperature (LCST) lower than 37° C. so that the polymer isdissolved by cooling the scaffold below cell culture temperatures. By“biodegradable,” it is meant that a polymer, once implanted and placedin contact with bodily fluids and/or tissues, will degrade eitherpartially or completely through chemical, biochemical and/or enzymaticprocesses. Non-limiting examples of such chemical reactions includeacid/base reactions, hydrolysis reactions, and enzymatic cleavage. Incertain non-limiting embodiments, the biodegradable polymers maycomprise homopolymers, copolymers, and/or polymeric blends comprising,without limitation, one or more of the following monomers: glycolide,lactide, caprolactone, dioxanone, and trimethylene carbonate. In othernon-limiting embodiments, the polymer(s) comprise labile chemicalmoieties, non-limiting examples of which include esters, ethers,anhydrides, polyanhydrides, or amides, which can be useful in, forexample and without limitation, controlling the degradation rate of thescaffold and/or the release rate of therapeutic agents from thescaffold. Alternatively, the polymer(s) may contain peptides orbiomacromolecules as building blocks which are susceptible to chemicalreactions once placed in situ. In one non-limiting example, the polymeris a polypeptide comprising the amino acid sequencealanine-alanine-lysine, which confers enzymatic lability to the polymer.

The polymer components may be selected so that they degrade in situ on atimescale that is similar to an expected rate of production of ECMscaffold by the cells seeded onto the template scaffold. Polyesters andpolyurethanes that incorporate hydrophilic groups such as ester or ethergroups are examples of polymer compositions that would degrade overtime. Examples of suitable polyesters include polylactic acid (PLA),polyglycolides (polyglycolic acid, PGA), polycapro-lactone (PCL),polydioxanone, polyhydroxyalkanoates (PHA), poly(lactic-co-glycolicacid) (PLGA), etc. Non-limiting examples of useful in situ degradationrates include between one week and one year or increments therebetweenfor instance, between one week and 10 months, and between one month andsix month. In the context of the present disclosure, it is desirablethat the synthetic polymer components degrade within one month, or less,so that aligned collagen and other ECM constituents replace the originalpolymeric components as rapidly as possible.

The polymeric components used to make the scaffold are biocompatible. By“biocompatible,” it is meant that a polymer compositions and its normaldegradation in vivo products are cytocompatible and are substantiallynon-toxic and non-carcinogenic in a patient within useful, practicaland/or acceptable tolerances. By “cytocompatible,” it is meant that thepolymer can sustain a population of cells and/or the polymercomposition, device, and degradation products, thereof are not cytotoxicand/or carcinogenic within useful, practical and/or acceptabletolerances. For example, the polymer when placed in a human cornea stemcell or functional keratocyte cell culture does not adversely affect theviability, growth, adhesion, and number of cells. In one non-limitingembodiment, the compositions, and/or devices are “biocompatible” to theextent they are acceptable for use in a human veterinary patientaccording to applicable regulatory standards in a given jurisdiction. Inanother example the biocompatible polymer, when implanted in a patient,does not cause a substantial adverse reaction or substantial harm tocells and tissues in the body, for instance, the polymer composition ordevice does not cause necrosis or an infection resulting in harm totissues from the implanted scaffold. In the context of the presentapplication, the original polymer composition is typically completely orsubstantially degraded when implanted.

The mechanical properties of a biodegradable scaffold can be optimizedto mimic native tissue at the site of implantation. In certainnon-limiting embodiments, the mechanical properties of the scaffold areoptimized similar to or identical to that of native soft tissue, such asfascia, connective tissue, blood vessel, muscle, tendon, fat, etc. Inone non-limiting embodiment, the biodegradable scaffold comprises athermoplastic polymer. The mechanical properties of the scaffold alsomay be optimized to be suitable for surgical handling. In onenon-limiting embodiment, the scaffold is flexible. In another, thescaffold is foldable and can be delivered to the site by minimallyinvasive methods.

The physical and/or mechanical properties of the biodegradable scaffoldcan be optimized by any useful method. Because the polymer compositionsare deposited by electrodeposition, more typically by electrospinning,the polymer structure is optimized for that application. Variables thatcan be optimized include without limitation, the extent of physicalcross-linking in a network comprising polymeric components, the ratio ofpolymeric components within the network, the distribution of molecularweight of the polymeric components, and the method of processing thepolymers. Polymers are typically semicrystalline and their physicalproperties and/or morphology are dependent upon a large number offactors, including monomer composition, polydispersity, averagemolecular weight, cross-linking, and melting/crystallization conditions.For example, flow and/or shear conditions during cooling orelectrodeposition of a polymer melt are known to affect formation ofcrystalline structures in the composition. In one non-limitingembodiment, the scaffold comprises a polymeric component that providesstrength and durability to the scaffold, yet is so that the mechanicalproperties of the scaffold are similar to the native tissue surroundingthe wound or site in need of tissue regeneration.

The polymeric component can be any useful biocompatible, biodegradableand synthetic polymer material. In certain non-limiting embodiments, thesynthetic polymeric component comprises a thermoplastic biodegradableelastomer. In another the polymeric component comprises aphase-separated biodegradable elastomer with degradable soft and/or hardsegments. In yet another non-limiting embodiment, the syntheticpolymeric component comprises any hydrolytically, chemically,biochemically, and/or proteolytically labile group, non-limitingexamples of which include an ester moiety, amide moiety, anhydridemoiety, specific peptide sequences, and generic peptide sequences.

In certain non-limiting embodiments, the polymeric component is abiodegradable polyurethane polymer. In one example, the syntheticpolymeric component is a linear segmented poly(urethane urea) copolymer,where the copolymer comprises alternating blocks of “soft” and “hard”segments. In one non-limiting embodiment, the soft segment is apolyether or polyester (e.g., polycaprolactone), which may have a glasstransition temperature (temperature at which a reversible change occursin an amorphous material, such as glass or an amorphous polymer, or inamorphous portions of a partially crystalline polymer from, or to, aviscous or rubbery condition to a hard or relatively brittle one) belowthe use temperature. As used herein, the “use temperature” or likephrases refers to the temperature at which the scaffolding is maintainedafter implantation, namely the body temperature of a patient, such as37° C. for a human patient or typical cell culture.

In another non-limiting embodiment, the soft segment comprises amultiblock copolymer in which one or more segments are polyester. In onenon-limiting embodiment, a pre-polymer is formed by reacting butyldiisocyanate with polycaprolactone diol and then further reacting thepre-polymer with a chain extender, such as butyl diamine and specificpeptide sequences (e.g., alanine-alanine-lysine).

The polymeric component can be prepared by any useful method. Accordingto one non-limiting embodiment, the polymeric component comprises abiodegradable polymeric portion, an isocyanate derivative, and a diaminechain extender. In one non-limiting example, formation of the polymericcomponent comprises at least two steps. In the first step, a pre-polymeris formed, for example in one non-limiting embodiment, the pre-polymercomprises an isocyanate-terminated polymer, which is formed by reactinga biodegradable polymer with an isocyanate derivative. In the secondstep, the pre-polymer can be further reacted to form chemical bondsbetween pre-polymer molecules. For example, the isocyanate-terminatedpre-polymer is reacted with a diamine chain extender, which reacts withthe isocyanate moiety to form chemical bonds between pre-polymermolecules. In another non-limiting example, the isocyanate-terminatedpre-polymer is reacted with a diol chain extender, which reacts with theisocyanate moiety. As used herein, an “isocyanate derivative” is anymolecule or group that is terminated by the moiety —N═C═O. Isocyanatederivates also include, without limitation, monoisocyanates andpolyisocyanates, such as diisocyanates and triisocyanates. In onenon-limiting embodiment, the isocyanate derivative is1,4-diisocyanatobutane.

Preparation of polymeric components may include other steps, including,for example and without limitation, catalytic steps, purification steps,and separation steps. The polymeric component described herein comprisesone or more biodegradable, biocompatible polymers. The biodegradablepolymers may be, without limitation, homopolymers, copolymers, and/orpolymeric blends. The polymer(s) may comprise, without limitation, oneor more of the following monomers: glycolide, lactide, caprolactone,dioxanone, and trimethylene carbonate. In one non-limiting embodiment,the polymer comprises a polycaprolactone. In another embodiment, thepolymer comprises a polycaprolactone diol. In yet another embodiment,the polymer comprises a triblock copolymer comprising polycaprolactone,poly(ethylene glycol), and polycaprolactone blocks.

As used herein, a “chain extender” is any molecule or group that reactswith an active group, such as, without limitation, an isocyanatederivative, to extend chains of polymers. Non-limiting examples ofuseful chain extenders are diamines and diols. In one non-limitingembodiment, the chain extender is a diamine that allows for extendingthe chain of the pre-polymer, such as putrescine (1,4-diaminobutane). Inanother non-limiting embodiment, the diamine is lysine ethyl ester. Inyet another non-limiting embodiment, the diamine is a peptide fragmentcomprising two or more amino acids, for example and without limitation,the peptide fragment alanine-alanine-lysine, which can be cleavedenzymatically by elastase. In one non-limiting embodiment, the chainextender is a diol that allows for extending the chain of thepre-polymer, such as 1,4-butane diol.

In one non-limiting embodiment, the polymeric component comprises abiodegradable poly(ester urethane) urea elastomer (PEUU). Onenon-limiting example of a PEUU is an polymer made from polycaprolactonediol (MW 2000) and 1,4-diisocyanatobutane, using a diamine chainextender, such as putrescine. The PEUU copolymer can be prepared by atwo-step polymerization process whereby polycaprolactone diol (MW 2000),1,4-diisocyanatobutane, and diamine are combined in a 2:1:1 molar ratio.In the first step, to form the pre-polymer, a 15 wt % solution of1,4-diisocyanatobutane in DMSO (dimethyl sulfoxide) is stirredcontinuously with a 25 wt % solution of polycaprolactone diol in DMSO.Then, stannous octoate is added and the mixture is allowed to react at75° C. for 3 hours. In the second step, the pre-polymer is reacted witha diamine to extend the chain and to form the polymer. For example andwithout limitation, the diamine putrescine is added drop-wise whilestirring and allowed to react at room temperature for 18 hours. Inanother non-limiting embodiment, the diamine is lysine ethyl ester,which is dissolved in DMSO with triethylamine, added to the pre-polymersolution, and allowed to react at 75° C. for 18 hours. After the twostep polymerization process, the polymer solution is precipitated indistilled water. Then, the wet polymer is immersed in isopropanol forthree days to remove any unreacted monomers. Finally, the polymer isdried under vacuum at 50° C. for 24 hours.

In another non-limiting embodiment, the polymeric component comprises apoly(ether ester urethane) urea elastomer (PEEUU). In one non-limitingexample, the PEEUU is made by reacting polycaprolactone-b-polyethyleneglycol-b-polycaprolactone triblock copolymers with1,4-diisocyanatobutane and putrescine. PEEUU may be obtained, forexample and without limitation, by a two-step reaction using a 2:1:1reactant stoichiometry of 1,4-diisocyanatobutane: triblockcopolymer:putrescine. In a further non-limiting example, the triblockpolymer is prepared by reacting poly(ethylene glycol) and ε-caprolactonewith stannous octoate at 120° C. for 24 hours under a nitrogenenvironment. The triblock copolymer may be washed with ethyl ether andhexane, then dried in a vacuum oven at 50° C. In the first step to formthe pre-polymer, a 15 wt % solution of 1,4-diisocyanatobutane in DMSO isstirred continuously with a 25 wt % solution of triblock copolymer inDMSO. Stannous octoate is then added and the mixture is allowed to reactat 75° C. for 3 hours. In the second step, putrescine is added drop-wiseunder stirring to the pre-polymer solution and allowed to react at roomtemperature for 18 hours. The PEEUU polymer solution is thenprecipitated with distilled water. The wet polymer is immersed inisopropanol for 3 days to remove unreacted monomer and dried undervacuum at 50° C. for 24 hours.

In another non-limiting embodiment, the polymer composition has a LowerCritical Solution Temperature (LCST) below 37° C. such that the polymerstructure solubilized at temperatures below cell culture temperatures.This facilitates removal of the original copolymer in favor of thealigned collagen scaffold formed by the corneal keratocytes. The LCSTmay preferably be between 20° C. and 35° C. in order to minimally affectECM structures formed by the cells in culture, but the In one example,the polymer composition comprises pNIPAAM (poly N-isopropyl acrylamide).

As described in U.S. Pat. No. 5,262,055, incorporated herein byreference in its entirety, thermosensitive polymers may be made up ofmonomers or mixtures of such monomers polymerizable by free radical orionic initiation which results in polymers having LCST in aqueoussystems between 15° C. and 35° C. Suitable are the N-alkyl orN,N-dialkyl substituted acrylamides or methacrylamides of the formula:

where R is hydrogen or methyl, R¹ is a member selected from the groupconsisting of lower alkyl and lower alkoxyalkyl and R² is a memberselected from the group consisting of hydrogen, lower alkyl and loweralkoxyalkyl with the proviso that R¹ and R² can combine as an alkylene—(CH₂)_(n)— chain to form a N-cyclic structure where n is an integer of4 to 6. n is preferably 5. By lower alkyl or alkoxy is meant a straightor branched carbon chain containing from one to eight carbon atoms andpreferably from one to five carbon atoms. Mixtures of one or more of theabove monomers may also be utilized as temperature sensitive components.

Examples of such temperature sensitive monomers are those selected fromthe group consisting of N-isopropylacrylamide [“NiPAAm”],N,N-diethylacrylamide, N-acryloylpiperidine, N-methylmethacrylamide,N-ethylmethacrylamide, N-n-propylacrylamide andN-(3′-methoxypropyl)acrylamide. The preferred temperature sensitivemonomers are the lower alkyl acrylamides which are selected from thegroup consisting of N-isopropylacrylamide, N,N-diethylacrylamide andN-n-propylacrylamide.

United States Patent Publication No. 20080096975 A1, incorporated hereinby reference for its technical disclosure, describes a useful copolymercomprising poly NIPAAM, n-hydroxyl succinimide, acrylic acid and apolyester macromer. According to one embodiment, the copolymer comprisesan N-isopropylacrylamide residue (an N-isopropylacrylamide monomerincorporated into a polymer), one or both of an acrylic acid residue anda methacrylic acid residue and an acrylic residue having anamine-reactive group. The copolymer comprises a polyester linkage in itsbackbone. According to one non-limiting embodiment, the copolymer isprepared from at least five components: N-isopropylacrylamide or anN-alkyl acrylamide in which the alkyl is methyl, ethyl, propyl,isopropyl or cyclopropyl, acrylic acid and/or methacrylic acid, anacrylic monomer optionally having an amine-reactive group (such asacrylic N-hydroxysuccinimide ester) and a polyester macromer. Forexample and without limitation, the polyester macromer is a polylactidemacromer, comprising hydroxyethyl methacrylate residues and varyingnumbers of lactide units/residues. In another non-limiting example, thepolyester macromer is a poly(trimethylene carbonate macromer),comprising hydroxyethyl methacrylate residues and varying numbers oftrimethylene carbonate units/residues. Each component contributes to thedesired physical properties of the hydrogel to form a distinct structureat a higher temperature and to solubilize at a lower temperature belowthe LCST of the composition. The amine-reactive component of thecopolymer (for instance, acrylic N-hydroxysuccinimide ester) binds toamine-containing compounds including bioactive or biocompatiblematerials or factors. The composition of each component in the hydrogeldetermines the lower critical solution temperature (LCST) of thehydrogel. At a temperature less than the LCST, the hydrogel flows easilyand can be injected into the desired shape. When the temperature isincreased above the LCST, the hydrogel solidifies and retains the shape.Once solidified, the hydrogel is highly flexible and relatively strongat physiological temperature.

According to one embodiment, polyester component within the macromerintroduces the degradability and hydrophobicity of the copolymer. Forcomplete removal of the copolymer, the copolymer includeshydrolytically-cleavable bonds that results in soluble, non-toxicby-products, even above the LCST of the non-degraded copolymer. Once thecopolymer is degraded, the LCST is above physiological temperature,which results in dissolution of the degraded hydrogel and clearance ofthe degraded components.

To facilitate the hydrolysis of the copolymer, according to oneembodiment, the backbone of the polymer comprises biodegradable esterlinkages, for example and without limitation, from 1% to 10% of thelinkages of the copolymer backbone. The polymer may comprise a polyestermacromer, for example and without limitation, a polyester macromercomprising hydroxyethyl methacrylate and lactide residues. In oneembodiment, the ratio of hydroxyethyl methacrylate and lactide residuesin the polyester macromer is from 1:2 to 1:8, in another, from 1:1 to1:10, such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10. Inanother non-limiting example, the polyester macromer compriseshydroxyethyl methacrylate and trimethylene carbonate residues. In oneembodiment, the ratio of hydroxyethyl methacrylate and trimethylenecarbonate residues in the polyester macromer ranges from 1:1 to 1:10,1:2 to 1:5 or any increment within those ranges, including 1:1, 1:2,1:3, 1:4, 1:4.2, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10. Table 1 providesexemplary structures (excerpted from United States Patent PublicationNo. 20080096975 A1)

TABLE 1 Composition of Poly(NIPAAm-co-NHS-co-AAc-co-HEMAPLA) PolymerFeed ratio Composition* P(NIPAAm-co-AAc-co-NHS-co- 85/6/5/485/6.7/3.9/4.4 HEMAPLA2.1**) P(NIPAAm-co-AAc-co-NHS-co- 85/6/5/485/6.9/4.0/4.1 HEMAPLA3.9**) P(NIPAAm-co-AAc-co-NHS-co- 85/6/5/485/6.9/3.8/4.3 HEMAPLA7.0**) P(NIPAAm-co-AAc-co-NHS-co- 80/6/5/980/7.5/4.2/8.3 HEMAPLA2.1**) P(NIPAAm-co-AAc-co-NHS-co- 80/6/5/980/7.0/4.4/8.6 HEMAPLA3.9**) P(NIPAAm-co-AAc-co-NHS-co- 75/6/5/1475/7.3/4.7/13.0 HEMAPLA2.1**) P(NIPAAm-co-AAc-co-NHS-co- 75/6/5/1475/6.3/4.9/13.8 HEMAPLA3.9**) P(NIPAAm-co-AAc-co-NHS-co- 80/11/5/480/11.4/4.2/4.4 HEMAPLA2.1**) P(NIPAAm-co-AAc-co-NHS-co- 80/11/5/480/10.6/6.2/3.2 HEMAPLA3.9**) *Determined by H1-NMR. **Lactide units

In general, the aligned, biodegradable scaffold described herein may bemade using any useful method, including one to the many common processesknown in the polymer and textile arts. Any method of forming alignedpolymeric fibers may be used to prepare the structures described herein.Spinnerette and extrusion methods such as wet, dry, melt and gelspinning may be used, depending on the physical properties of thepolymer composition. Electrodeposition is one useful method of preparingsmall fibers useful in producing biological scaffolds. Aligned fibersmay be formed by electrospinning, a modification of theelectrodeposition method.

In other non-limiting embodiments, electrospinning is used to fabricatethe scaffold. The process of electrospinning involves placing apolymer-containing fluid (for example, a polymer solution, a polymersuspension, or a polymer melt) in a reservoir equipped with a smallorifice, such as a needle or pipette tip and a metering pump. Oneelectrode of a high voltage source is also placed in electrical contactwith the polymer-containing fluid or orifice, while the other electrodeis placed in electrical contact with a target (such as a collectorscreen or rotating mandrel). During electrospinning, thepolymer-containing fluid is charged by the application of high voltageto the solution or orifice (for example, about 3-15 kV) and then forcedthrough the small orifice by the metering pump that provides steadyflow. While the polymer-containing fluid at the orifice normally wouldhave a hemispherical shape due to surface tension, the application ofthe high voltage causes the otherwise hemispherically shapedpolymer-containing fluid at the orifice to elongate to form a conicalshape known as a Taylor cone. With sufficiently high voltage applied tothe polymer-containing fluid and/or orifice, the repulsive electrostaticforce of the charged polymer-containing fluid overcomes the surfacetension and a charged jet of fluid is ejected from the tip of the Taylorcone and accelerated towards the target, which typically is biasedbetween −2 to −10 kV. Optionally, a focusing ring with an applied bias(for example, 1-10 kV) can be used to direct the trajectory of thecharged jet of polymer-containing fluid. As the charged jet of fluidtravels towards the biased target, it undergoes a complicated whippingand bending motion. If the fluid is a polymer solution or suspension,the solvent typically evaporates during mid-flight, leaving behind apolymer fiber on the biased target. If the fluid is a polymer melt, themolten polymer cools and solidifies in mid-flight and is collected as apolymer fiber on the biased target. As the polymer fibers accumulate onthe biased target, a non-woven, porous mesh is formed on the biasedtarget.

The properties of the electrospun scaffolds can be tailored by varyingthe electrospinning conditions. For example, when the biased target isrelatively close to the orifice, the resulting electrospun mesh tends tocontain unevenly thick fibers, such that some areas of the fiber have a“bead-like” appearance. However, as the biased target is moved furtheraway from the orifice, the fibers of the non-woven mesh tend to be moreuniform in thickness. Moreover, the biased target can be moved relativeto the orifice. In certain non-limiting embodiments, the biased targetis moved back and forth in a regular, periodic fashion, such that fibersof the non-woven mesh are substantially parallel to each other(aligned). When this is the case, the resulting non-woven mesh may havea higher resistance to strain in the direction parallel to the fibers,compared to the direction perpendicular to the fibers.

The target can also be a rotating mandrel. In this case, the propertiesof the non-woven mesh may be changed by varying the speed of rotation.The properties of the electrospun scaffold may also be varied bychanging the magnitude of the voltages applied to the electrospinningsystem. In one non-limiting embodiment, the electrospinning apparatusincludes an orifice biased to 12 kV, a target biased to −7 kV, and afocusing ring biased to 3 kV. Moreover, a useful orifice diameter is0.047″ (I.D.) and a useful target distance is about 23 cm.

Other electrospinning conditions that can be varied include, for exampleand without limitation, the feed rate of the polymer solutions, thedistance from the source to the target, the solution concentrations, andthe polymer molecular weight. Sheets of aligned polymer fibers areprepared by using a rotating mandrel as a target. The mandrel is notnecessarily circular in cross-section (and thus cylindrical), buttypically is cylindrical. The mandrel may have any diameter and can beused to prepare sheets having a width that is up to the axial length ofthe mandrel and a length that is the circumference of the mandrel. Onceformed, the aligned fiber sheet can be cut off of the mandrel to form aplanar sheet. The thickness of the sheet will depend on the number ofturns the polymer fibers make about the mandrel during electrospinningand to some extent the thickness of the fibers.

In order to facilitate continuous formation of multiple layers, as analternative to electrospinning, the fibers and cells can beelectrodeposited layer-by-layer. In this embodiment, the target hasalternate grounds arranged such that fibers of each alternate layer aredeposited at a different angle, e.g., 90° or 45° with respect to fibersof another layer. In this instance, fibers are deposited in a firstorientation using a first ground. Cells are deposited by electrosprayingand then fibers are deposited in a second orientation, not parallel tothe first orientation using a second ground.

In the Examples below, a PEUU sheet of aligned fibers is formed about amandrel that is 2 cm long (axial) and 20 cm in diameter. The conditionsused to obtain an aligned fiber structure that yielded the bestappearance, and therefore was determined best for the experimentationdescribed below was 2000 RPM for a 5% PEUU solution inhexafluoroisopropanol (HFIP). It should be noted that for everydifferent polymer composition, choice of solvent and target, the optimalelectrospinning conditions may be obtained by varying target velocityand polymer solution composition to obtain a nonwoven structure withaligned fibers. This is well within the ability of one of ordinary skillin the art. Non-limiting examples of useful range of high-voltage to beapplied to the polymer suspension is from 0.5 to 30 kV, from 5 to 25 kV,and from 10 to 15 kV. Useful range of concentrations for the polymericcomponents include, without limitation, from 1 wt % to 15 wt % includingincrements therebetween, for example from 4 wt % to 10 wt %, and from 6wt % to 8 wt %. In the examples below, HFIP is used as a solvent.

A number of other methods exist by which sheets or layers of alignedfibers can be electrodeposited. For example, an electrodeposition devicehaving two, split electrodes may be utilized as described in Li, D., etal. (Electrospinning Nanofibers as Uniaxially Aligned Arrays andLayer-by-Layer Stacked Films, Adv. Mater. 2004 16 361).

Cells may be integrated with the scaffold using a variety of methods.For example, the scaffold may be submersed in an appropriate medium (asolution capable of maintaining the viability of or supporting growth ofthe cells) for the cells, and then directly exposed to the cells. Thecells are allowed to adhere to the scaffold and optionally proliferateon the surface and interstices of the scaffold. Multiple layers of thescaffold may be stacked, and the cells propagated under suitable cultureconditions prior to further processing. From 2 to 200 layers may bestacked, though media may not adequately diffuse among the layers if toomany layers are stacked. As such, from 2-20 layers are preferablystacked, including increments therebetween, including 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 layers. Preferably,for preparation of cornea stromal material, the layers are orthogonallyaligned, meaning the fiber orientation in the layers are perpendicularto (including approximately or substantially perpendicular to) the fiberorientation in adjacent layers.

In another embodiment, the cells of interest are dissolved into anappropriate solution (e.g., a growth medium or buffer) and then sprayedonto a biodegradable scaffold while the scaffold is being formed byelectrospinning. While pressure spraying (that is, spraying cells from anozzle under pressure) is contemplated herein, in certain non-limitingembodiments, the cells are electrosprayed onto the non-woven mesh duringelectrospinning. As described herein, electrospraying involvessubjecting a cell-containing solution with an appropriate viscosity andconcentration to an electric field sufficient to produce a spray ofsmall charged droplets of solution that contain cells. United StatesPatent Publication No. 20080109070, incorporated herein by reference inits entirety for its technical disclosure, describes methods ofelectrospraying cells. These different conditions include sprayingalone, spraying onto a target charged at −15 kV, spraying onto a targetcharged at −15 kV with PEUU electro spinning, electrospraying at 10 kVonto a target charged at −15 kV, and electrospraying at 10 kV onto atarget charged at −15 kV with PEUU electrospinning. In contrast topressurized spraying, electrospraying cells using the methods describedherein did not significantly affect cell viability or proliferation. Itis preferred that the distance between the polymer composition andmandrel is sufficient, and the relative orientation of the polymer andcell spraying nozzle is such that the cells do not contact polymersolution containing the solvent (e.g. HFIP).

In the context of the present disclosure, the cells to be deposited ontothe scaffold are cells that are native to the tissue to be engineered,or precursors thereof (e.g., stem cells). As an example, for preparationof an engineered corneal stroma structure, it is most desirable todeposit cells that are corneal keratocytes, keratocytes that thatproduce corneal stroma ECM material (such as keratocytes derived fromadipose stem cells, or stem cells that differentiate in culture tocorneal keratocytes or keratocytes that that produce suitable cornealstroma ECM material). Corneal stroma ECM material is an ECM thatcomprises aligned collagen, keratan sulfate and keratocan.

One or more of therapeutic agents can be introduced into the scaffold byany useful method, such as, without limitation absorption, adsorption,deposition, admixture with a polymer composition used to manufacture thescaffold and linkage of the agent to a component of the scaffold. Itshould be noted that the active agents can be added to the templatescaffold and/or to the product ECM scaffold formed within the templatescaffold. In one non-limiting example, the therapeutic agent isintroduced into a backbone of a polymer used in the template scaffold.By adding the therapeutic agent to the polymer itself, the rate ofrelease of the therapeutic agent may be controlled by the rate ofpolymer degradation. During an electrospinning process, the therapeuticagent can be electrosprayed onto the polymer being spun. In yet anothernon-limiting example, the therapeutic agent is introduced into thescaffold after the product ECM scaffold is formed. For instance, thescaffold may be “loaded” with therapeutic agent(s) by using staticmethods. For instance, the scaffold can be immersed into a solutioncontaining the therapeutic agent permitting the agent to absorb intoand/or adsorb onto the scaffold. The scaffold may also be loaded byusing dynamic methods. For instance, a solution containing thetherapeutic agent can be perfused or electrodeposited into the scaffold.

Therapeutic agents within the template and product ECM scaffold can beused in any number of ways. In one non-limiting embodiment, atherapeutic agent is released from the scaffold. For example and withoutlimitation, anti-inflammatory drugs are released from the scaffold todecrease an immune response. When the ECM product scaffold is implantedin a patient's cornea, it may be desirable to include ananti-inflammatory agent and/or an antibiotic. Non-limiting examples ofsuitable antibiotics include: ciprofloxacin, norfloxacin, afloxacin,levofloxacin, gentamicin, tobramycin, neomycin, erythromycin,trimethoprim sulphate, and polymyxin B. Steroidal anti-inflammatoriesare useful, but not preferred because they cause corneal thinning.Non-steroidal anti-inflammatories (NSAIDs) suitable for ocular use arepreferred and include, without limitation: nepafenac (for example andwithout limitation, Nevenac 0.1%, nepafenac ophthalmic suspension, AlconLaboratories, Inc.), ketorolac tromethamine (for example and withoutlimitation, Acular LS 0.4%, ketorolac tromethamine ophthalmicsuspension, Allergan, Inc.), acetaminophen and bromfenac (for exampleand without limitation, Xibrom 0.09%, bromfenac ophthalmic suspension,Ista Pharmaceuticals). In another non-limiting embodiment, a therapeuticagent is intended to substantially remain within the scaffold. Forexample and without limitation, chemoattractants are maintained withinthe scaffold to promote cellular migration and/or cellular infiltrationinto the scaffold.

In a non-limiting embodiment, at least one therapeutic agent is added tothe product ECM scaffold before it is implanted in the patient.Generally, the therapeutic agents include any substance that can becoated on, embedded into, absorbed into, adsorbed to, or otherwiseattached to or incorporated onto or into the biodegradable scaffold thatwould provide a therapeutic benefit to a patient. Non-limiting examplesof such therapeutic agents include antimicrobial agents, growth factors,emollients, retinoids, and steroids. Each therapeutic agent may be usedalone or in combination with other therapeutic agents.

In certain non-limiting embodiments, the therapeutic agent is a growthfactor, such as a neurotrophic or angiogenic factor, which optionallymay be prepared using recombinant techniques. Non-limiting examples ofgrowth factors include basic fibroblast growth factor (bFGF), acidicfibroblast growth factor (aFGF), vascular endothelial growth factor(VEGF), hepatocyte growth factor (HGF), insulin-like growth factors 1and 2 (IGF-1 and IGF-2), platelet derived growth factor (PDGF), stromalderived factor 1 alpha (SDF-1 alpha), nerve growth factor (NGF), ciliaryneurotrophic factor (CNTF), neurotrophin-3, neurotrophin-4,neurotrophin-5, pleiotrophin protein (neurite growth-promoting factor1), midkine protein (neurite growth-promoting factor 2), brain-derivedneurotrophic factor (BDNF), tumor angiogenesis factor (TAF),corticotrophin releasing factor (CRF), transforming growth factors α andβ (TGF-α and TGF-β), interleukin-8 (IL-8), granulocyte-macrophage colonystimulating factor (GM-CSF), interleukins, and interferons. Commercialpreparations of various growth factors, including neurotrophic andangiogenic factors, are available from R & D Systems, Minneapolis,Minn.; Biovision, Inc, Mountain View, Calif.; ProSpec-Tany TechnoGeneLtd., Rehovot, Israel; and Cell Sciences®, Canton, Mass.

In certain non-limiting embodiments, the therapeutic agent is anantimicrobial agent, such as, without limitation, isoniazid, ethambutol,pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones,ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin,dapsone, tetracycline, erythromycin, ciprofloxacin, doxycycline,ampicillin, amphotericin B, ketoconazole, fluconazole, pyrimethamine,sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone,paromomycin, diclazaril, acyclovir, trifluorouridine, foscarnet,penicillin, gentamicin, ganciclovir, iatroconazole, miconazole,Zn-pyrithione, and silver salts such as chloride, bromide, iodide andperiodate.

In certain non-limiting embodiments, the therapeutic agent is ananti-inflammatory agent, such as, without limitation, an NSAID, such assalicylic acid, indomethacin, sodium indomethacin trihydrate,salicylamide, naproxen, colchicine, fenoprofen, sulindac, diflunisal,diclofenac, indoprofen, sodium salicylamide; an anti-inflammatorycytokine; an anti-inflammatory protein; a steroidal anti-inflammatoryagent; or an anti-clotting agents, such as heparin.

In certain non-limiting embodiments, the therapeutic agent comprisescells that are added to the biodegradable scaffold before or at the timeof implantation. The cells may be cells used to transform the templatescaffold to the product ECM scaffold. Because the cells are to beimplanted in the cornea of a patient, though the eye isimmune-privileged, it may be preferred to use only autologous cells. Theuse of autologous functional keratocytes implies that the functionalkeratocytes or corneal stroma stem cells are obtained from the cornea ofthe implant patient (as corneal stem cells or keratocytes), and then thecells are propagated and seeded onto the template scaffold. As such, itmay be that the only time this strategy is used is where the patient hassuch substantial scarring to the cornea that removal of a portion of thepatient's stroma for ex vivo expansion of cells would be acceptable tothe patient.

“Functional keratocytes” are cells that have the ability to deposit anorganized transparent ECM scaffold, but which may or may not bekeratocytes per se as they exist in the human body, or other animal,with precisely the same phenotypic markers as natively found in thehuman body. They may be differentiated cells that are not literallykeratocytes, but cells exhibiting the ability to function as keratocytesin their ability to deposit organized, transparent ECM includingcollagen, keratan sulfate and keratocan. As indicated below, the cellsdifferentiated from hCSSCs and ADSCs in KDM are considered to befunctional keratocytes, whether or not they are strictly keratocytes asfound in the human body. Nevertheless, “functional keratocytes” includeskeratocytes as they naturally exist.

As mentioned above, virtually any cell, human or non-human can be usedto seed the template scaffold, so long as it can be used to prepare auseful product ECM. For many tissues, the choice of cells is lesscritical than in the case of cornea repair or correction. Where thefinal scaffold product is decellularized, the cells used to seed thetemplate scaffold do not have to be autologous. For instance allogeneichuman corneal stem cells can be obtained as described below, for examplefrom donors where the cornea is not suitable for direct transplantation.As also described below, xenogeneic corneal stem cells also may be usedbecause they may actually form a product ECM scaffold that is moresuitable for human implantation than a scaffold formed from humancorneal keratocytes differentiated from human corneal stem cells exvivo. This would be a matter of optimization using the methods describedherein.

The product ECM scaffold can be sterilized, and typically decellularizedby any of a number of standard methods without loss of its ability toinduce endogenous tissue growth. The material may be decellularized byrepeated freeze-thaw cycles and then washed to remove debris. In anotherexample, the material can be sterilized by propylene oxide or ethyleneoxide treatment, gamma irradiation treatment (0.05 to 4 mRad), gasplasma sterilization, peracetic acid sterilization, or electron beamtreatment. The scaffold can be disinfected by immersion in 0.1% (v/v)peracetic acid (σ), 4% (v/v) ethanol, and 96% (v/v) sterile water for 2h. The ECM material is then washed twice for 15 min with PBS (pH=7.4)and twice for 15 min with deionized water. Product ECM preparations canbe considered to be “decellularized”, meaning the cells have beenremoved from the source tissue through processes described herein andknown in the art.

In one non-limiting embodiment, the cells used to prepare the scaffoldmay be genetically modified cells that are capable of expressing atherapeutic substance, such as a growth factor, or even one or morespecific ECM constituents. Cells can be modified by any usefulrecombinant method in the art. For example and without limitation, thetherapeutic agent is a growth factor that is released by cellstransfected with cDNA encoding for the growth factor. Therapeuticsagents that can be released from cells include, without limitation, aneurotrophic factor, such as nerve growth factor, brain-derivedneurotrophic factor, neurotrophin-3, neurotrophin-4, neurotrophin-5, andciliary neurotrophic factor; a growth factor, such as basic fibroblastgrowth factor (bFGF), acidic fibroblast growth factor (aFGF), vascularendothelial growth factor (VEGF), hepatocyte growth factor (HGF),insulin-like growth factors (IGF), platelet derived growth factor(PDGF), transforming growth factor-beta (TGF-β), pleiotrophin protein(neurite growth-promoting factor 1), and midkine protein (neuritegrowth-promoting factor 2); an anti-inflammatory cytokine; and ananti-inflammatory protein. The cells may be autologous, allogeneic, etc.

In addition to preparing and providing the product ECM scaffolds asdescribed above, methods of using such scaffolds are encompassed herein.Generally, the scaffold can be implanted by using any suitable medicalprocedure that facilitates use of the scaffold to provide a therapeuticbenefit. As used herein, the terms “implanted” and “implantation” andlike terms refer to an act of delivering a biodegradable elastomericscaffold to a site within the patient and of affixing the scaffold tothe site. The site of implantation in a patient typically is “at or neara site for wound healing or tissue generation or regeneration in thepatient,” meaning the scaffold-containing device is implanted in, on,onto, adjacent to or in proximity to a desired site of delivery tofacilitate healing and/or tissue generation or regeneration to repair aninjury or defect in the patient and/or to achieve a desired effect inthe patient, such as wound drainage. The delivery method may alsoinclude minimally invasive methods such as by catheter based technologyor by needle injection. The patient may be human or animal. The scaffoldmay be delivered by any surgical procedure, including minimally invasivetechniques, such as laparoscopic surgery, as well as invasive techniquessuch as thoracic surgery and fasciotomy. In certain non-limitingembodiments, the elastomeric scaffolds are used as surgical fabrics. Forexample and without limitation, the scaffold can be implanted in apatient during laparoscopic procedures to repair or to reinforce fasciaethat have been damaged or weakened. The elastomeric scaffolds can alsobe used to re-join organs that have been separated as a result ofsurgery, to treat hernias, and to promote the healing of surgicalincisions. The scaffold may be implanted alone or implanted inconjunction with surgical fasteners, such as sutures, staples,adhesives, screws, pins, and the like. Additionally, biocompatibleadhesives, such as, without limitation, fibrin-based glue) may be usedto fasten the scaffolds as well.

In other non-limiting embodiments, the biodegradable elastomericscaffolds may be used to promote healing of deep tissue wounds, such aspuncture wounds, bullet wounds, or wounds that result from the surgicalremoval of a substantial amount of tissue, such as in debridementprocedures or removal of tumors. In yet another non-limiting embodiment,the scaffold can be in the form of a powder or fine particles (forexample, formed by shredding a non-woven mesh formed the methodsdescribed herein), and is packed directly into the wound to provide amatrix on which the patient's cells may grow. In these situations, itmay be advantageous to derivatize the scaffold with therapeutic agents,such as antibiotics or growth factors, prior to insertion into thewound.

According to one non-limiting embodiment, the scaffold is a cornealstroma scaffold that is prepared as described above. In this embodiment,the product ECM scaffold is prepared and/or processed in a shape andsize suitable for insertion into the cornea as a stroma replacement orsupplement. Therefore the scaffold may replace scarred or damagedcorneal stroma tissue, or may be implanted in a refractive surgicalprocedure to change the refractivity of the cornea to correct refractiveimperfections in the eye.

This method comprises transplanting shaped discs of bioengineeredstromal tissue or decellularized ECM material into healthy eyes in orderto change the refractive power of the cornea and reduce the need forcontact lenses or glasses. It involves placing the tissue at the surface(onlay) or in a stromal pocket (inlay) near the front of the stroma. Theshape of the tissue results in altered corneal refraction correctingrefractive errors in the eye that require glasses. In a corneal inlaymethod, the scaffold material is placed within the cornea under aLASIK-style flap. Then in position, the implant changes the curvature ofthe cornea such that the front of the eye acts in the manner of amultifocal contact lens. This method is used in connection with the Vue+lens (Revision Optics, Lake Forest, Calif.). In another method, a lasercreates a tiny pocket in the cornea into which the scaffold is placed.This method is used in connection with the Flexivue Microlens (PresbiaCoöperatief, U.A., Amsterdam). In another embodiment, the scaffold isimplanted as an onlay by placing the scaffold just under the epitheliallayer on the front surface of the cornea. This method is used inconnection with the corneal onlay provided by Adventus Technology ofIrvine, Calif.

Examples Example 1—Use of hCSSCs to Produce Corneal Stroma Biomaterial

An orderly three dimensional (3-D) collagen fibril nano-construct thatmimics corneal stroma tissue was generated by employing a tissueengineering strategy. We demonstrated that aligned nanofibrous scaffoldprepared from poly(ester urethane) urea (PEUU) provided the topographiccues to regulate morphogenesis of human corneal stromal stem cells(hCSSCs), and initiate and guide self-organization of hCSSC-secretedcollagen fibrils into 3-D orderly construct. The yielded constructfeatures the uniform fiber diameter and interfibrillar spacing, whichare believed to be controlled by collagens and proteoglycans typifyinghuman corneal stromal tissue. These striking results represent animportant first step of a bottom-up strategy to bioengineer complexcollagen-based nano-biological construct for tissue repair andregenerative medicine.

Materials

The biodegradable Poly(ester urethane) urea (PEUU) was synthesized asfollows. First, 1,4-diisocyanatobutane and polycaprolactone-diol (PCL,M_(w)=2 kg/mol) were reacted in dimethyl sulfoxide (DSMO, AnhydrousGrade) for three hour at 75° C. with the aid of Tin 2-ethylhexanoateunder the protection of Ar₂ purge. After cooling down to roomtemperature, the oligomer solution was drop-wise added to1,4-diaminobutane under vigorous stirring. After 18 hour reaction atroom temperature, the polymer solution was precipitated in distilledwater. Then the precipitant was soaked in anhydrous 2-propanol foranother 48 hours to remove DMSO and unreacted monomers. The yieldedpolymer was incubated in anhydrous ethanol for another 24 hours toremove water, and then further dried under vacuum at 40° C. for one weekin order to remove water residual. The yielded product was a whiteelastomer.

Cell Culture

Donor human corneas not usable for transplantation were rinsed andincubated in 2.4 U/ml Dispase II (Roche Diagnostics, Pleasanton, Calif.)overnight at 4° C. Epithelial and endothelial cells were removed bydissection and debridement, and the stroma was minced into 2-mm cubes.Stroma was digested up to 3 hours at 37° C. in Dulbecco's modifiedEagle's medium (DMEM) containing 1 mg/ml collagenase type L(Sigma-Aldrich) and 0.2 mg/ml testicular hyaluronidase (Sigma-Aldrich).Primary stromal cells were cultured at 1×10⁴ per cm² in a humidifiedatmosphere containing 5% CO₂ in a medium (stem cell growth medium[SCGM]) modified from Jiang et al. (Pluripotency of mesenchymal stemcells derived from adult marrow. Nature 2002;418:41-49) containingDMEM/MCDB-201 (Sigma-Aldrich) with 2% fetal bovine serum (FBS) (HyClone,Logan, Utah), 10 ng/ml epidermal growth factor (Invitrogen Corporation,Carlsbad, Calif.), 10 ng/ml platelet-derived growth factor (PDGF-BB)(Sigma-Aldrich), 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenousacid (ITS) (Invitrogen), 1,000 units per ml leukemia inhibitory factor(LIF) (Chemicon International), ×1 linoleic acid-bovine serum albumin(LA-BSA), 0.1 mM ascorbic acid-2-phosphate, 10-8 M dexamethasone, 100IU/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin, and 1.25μg/ml amphotericin B (Sigma-Aldrich). When 80%-90% confluent, cells weretrypsinized and subcultured. Passage 6 cells were used for cell culture.

Cell Sorting

At passage four, trypsinized cells were incubated at 1.0×10⁶ cells perml in DMEM with 2% FBS and 5 μg/ml Hoechst 33342 dye (Molecular Probes,Inc.) for 90 minutes at 37° C. To inhibit ABCG2-mediated efflux ofHoechst dye, cells were preincubated for 20 minutes with 50 μg/mlverapamil (an inhibitor of multidrug resistance proteins)(Sigma-Aldrich) before Hoechst 33342 incubation. After staining, thecells were washed twice in Hanks' balanced salt solution (HBSS) with 2%FBS and then stored in cold HBSS with 2% FBS on ice Immediately beforesorting, 2 μg/ml propidium iodide (Sigma-Aldrich) was added to identifynonviable cells for flow cytometric analysis. Cells were analyzed on aMoFlo (DakoCytomation, Fort Collins, Colo.) high-speed cell sorter,using 350-nm excitation. Cells showing reduced fluorescence of both blue(670 nm) and red (450 nm), a “side population,” were collected. Deadcells stained with propidium iodide were omitted from the population.

SP Cell Culture, Cloning, and Differentiation

After sorting, SP cells were cultured in SCGM. At 80%-90% confluence,these cells were cloned by limiting dilution and subcultured at adensity of 1×10⁴ cells per cm². Cloned cells were used in all subsequentexperiments. To determine differentiation potential, cloned passage-18SP cells were incubated 2 weeks in Advanced D-MEM (InvitrogenCorporation) supplemented with fibroblast growth factor 2 (FGF2) and 10ng/ml (keratocyte differentiation medium [KDM]).

Antibodies used included anti-keratocan peptide antibody (Guan, J.;Fujimoto, K. L.; Sacks, M. S.; Wagner, W. R. Biomaterials 2005, 26,3961-3971), J19 monoclonal to keratan sulfate (Sigma-Aldrich),anti-collagen I (Sigma-Aldrich), anti-collagen V (Chemicon, Temecula,Calif.), anticollagen VI (Chemicon, Temecula, Calif.) forimmunostaining. For fluorescent staining, Alexa Fluor-488 anti-mouseIgG, Alexa Fluor-546 anti-rabbit IgG, and nuclear dye DAPI were obtainedfrom Molecular Probes (Eugene, Oreg.).

Scaffold Preparation

The oriented nanofibrous scaffolds were prepared by electrospinningtechnique. Briefly, PEUU was dissolved in hexafluoroisopropanol (HFIP)under mechanical stirring at room temperature. The obtained polymersolution was fed by syringe pump (Harvard Apparatus) into a steelcapillary (I.D.=0.047 inch) suspended on an aluminum wheel collectorwith 2-cm in width and 20-cm in thickness. A combination of twohigh-voltage generators (Gamma high Voltage Research) was employed witha high positive voltage (+10 kV) to charge the steel capillarycontaining polymer solution, and a high negative voltage (−5 kV) tocharge the aluminum wheel collector with 20 cm in diameter. The distancebetween the tip of the steel capillary and the top of the aluminum wheelcollector is 15 cm. The volume flow rate was set up as 1 ml/hr. The PEUUsolution was electrospun with 5.0 wt-% concentration and rotationalspeed is 2000 rpm. The yielded fibrous scaffold is approximate 200micron thick.

The cast film was prepared as a control to assess the influence ofartificial surface features on cell morphology and the result collagenself-organization. The 5.0 wt-% PEUU/HFIP solution was poured in Teflon′casting dish, where the solvent evaporated to yield semitransparent,mechanically robust films with prescribed thickness of 0.2˜0.3 mm. Allof the scaffolds were dried in vacuum oven at room temperature for oneweek in order to eliminate the solvent, e.g. HFIP, completely.

Cell Cultures

The scaffold was punched into round discs with 25-mm in diameter inorder to fit in 24-well culture plate. The discs were sterilized by UVexposure (254 nm) in cell culture hood for 20 minute each side. ThehCSSCs were statically seeded on the scaffolds at a density of 8.0×10⁴cells/cm², which were incubated in stem cell growth medium (SCGM)containing DMEM/MCDB-201 with 2% fetal bovine serum (FBS), 10 ng/mlDMEM/MCDB-201 with 2% fetal bovine serum (FBS), 10 ng/ml epidermalgrowth factor, 10 ng/ml platelet-derived growth factor (PDGF-BB), 5μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenous acid (ITS), 1,000units per ml leukemia inhibitory factor (LIF), ×1 linoleic acid-bovineserum albumin (LA-BSA), 0.1 mM ascorbic acid-2-phosphate, 10-8 Mdexamethasone, 100 IU/ml penicillin, 100 μg/ml streptomycin, 50 μg/mlgentamicin, and 1.25 μg/ml amphotericin B.

After three day incubation in SCGM, hCSSC was transferred intoketarocyte differentiation medium (KDM, (KDM; advanced-MEM (Invitrogen)with 10 ng/mL basic fibroblast growth factor (bFGF, Sigma-Aldrich), 0.1mM L-ascorbic acid-2-phosphate (A2P, Sigma-Aldrich), L-glutamine(1×GlutaMax™-1; Invitrogen), 50 μg/ml Gentamicin (Invitrogen), 100 μg/mlpenicillin (Mediatech, Inc.), which was changed twice one week for up to6 weeks.

Two-Photon Fluorescent Microscopy

Differentiated hCSSC morphologies were observed with Two-photonFluorescent microscope. Scaffolds were randomly chosen from 24-wellplate on day 14, 28, and 42. Scaffolds were washed with PBS and werestained with CellTracker™ Green CMFDA (5-chloromethylfluoresceindiacetate) (Invitrogen) for 10 minutes. Samples were observed undertwo-photon fluorescent microscope.

Electron Microscopy

The morphologies of the differentiated hCSSCs and their secretedextracellular matrix on the scaffold were investigated by ScanningElectron Microscope (SEM). The specimens were fixed in cold 2.5%glutaraldehyde (25% glutaraldehyde EM grade, Taab Chemical) in 0.1 M PBS(sodium chloride, potassium chloride, sodium phosphate dibasic,potassium phosphate monobasic, Fisher), pH=7.3. The specimens wererinsed in PBS, post-fixed in 1% Osmium Tetroxide (Osmium Tetroxidecrystals, Electron Microscopy Sciences) with 0.1% potassium ferricyanide(Potassium Ferricyanide, Fisher), dehydrated through a graded series ofethanol (30%-90%-Reagent Alcohol, Fisher, and 100%-Ethanol 200 Proof,Pharmco), and hexamethyldisilazane (HMDS). The yielded sample wasinvestigated at 5 kV by Jeol JSM-6330F Scanning Electron Microscope(SEM) equipped with a digital camera.

The internal microstructures of the yielded ECM were investigatedemploying Transmission Electron Microscope (TEM). The sample was cutparallel and perpendicular to the alignment direction of PEUU fibrousscaffolds, respectively, in order to assess the influence of scaffoldsurface features on the ECM organization. The specimens were fixed incold 2.5% glutaraldehyde (25% glutaraldehyde EM grade, Taab Chemical) in0.1M PBS (sodium chloride, potassium chloride, sodium phosphate dibasic,potassium phosphate monobasic, Fisher), pH=7.3. The specimens wererinsed in 1×PBS, post-fixed in 1% Osmium Tetroxide (Osmium Tetroxidecrystals, Electron Microscopy Sciences) with 0.1% potassium ferricyanide(Potassium Ferricyanide, Fisher), dehydrated through a graded series ofethanol (30%-90%—Reagent Alcohol, Fisher, and 100%—Ethanol 200 Proof,Pharmco) and embedded in Epon (Dodecenyl Succinic Anhydride, NadicMethyl Anhydride, Scipoxy 812 Resin and Dimethylaminomethyl, Energy BeamSciences). Semi-thin (300 nm) sections were cut on a Reichart Ultracut,stained with 0.5% Toluidine Blue (Toluidine Blue O and Sodium Borate,Fisher) and examined under the light microscope. Ultrathin sections (65nm) were stained with 2% uranyl acetate (Uranyl Acetate dihydrate,Electron Microscopy Sciences, and methanol, fisher) and 1%phosphotungstic acid (Sigma-Aldrich), pH 3.2. The sections were examinedand photographed at 80 kV on Jeol 1011 transmission electron microscopeequipped with a digital camera.

Gene Expression

RNA of the differentiated hCSSCs seeded on the PEUU scaffolds wasisolated using the RNeasy mini kit (Qiagen, Valencia, Calif.). RNA wastreated with DNAse I (Ambion) and was concentrated by alcoholprecipitation. RNA (200 ng) was transcribed to cDNA in a 50 μL reactioncontaining 1×PCR II buffer (Roche Applied Science, Indianapolis, Ind.),5 mM MgCl2, 200 μM dNTP mixture (Roche), 2.5 μM random hexamers(Invitrogen), 0.4 U RNase inhibitor, and 125 U SuperScript II reversetranscriptase (Invitrogen). Quantitative PCR of cDNA was performed usingassays containing fluorescent hybridization probes (TaqMan; AppliedBiosystems, Foster City, Calif.) or with direct dye binding (SYBR Green;Applied Biosystems) according to the manufacturer's instructions.Reactions were carried out on triplicate samples for 40 cycles of 15seconds at 95° C. and 1 minute at 60° C. after initial incubation at 95°C. for 10 minutes. Reaction volume was 20 μL. For TaqMan assays,reactions contained 1× Universal PCR Master Mix (Applied Biosystems), 1×gene mix, and 3.0 μL cDNA. For SYBR dye-based assays, the reactionscontained 1×PCR buffer (Applied Biosystems), 3 mM Mg²⁺, 200 μM dATP,dCTP, dGTP, and 400 μM dUTP, 0.025 U/mL AmpliTaq Gold polymerase, 1.6 μLcDNA and forward and reverse primers at optimized concentrations.Amplification of 18S rRNA was carried out for each cDNA as a qualitativeexternal control. A dissociation curve for each SYBR-based reaction wasgenerated on a real-time thermocycler (Gene-Amp ABI Prism 7700 SequenceDetection System; Applied Biosystems) to confirm the absence ofnonspecific amplification. Amplification of 18S rRNA was performed foreach cDNA (in triplicate) for normalization of RNA content. RelativemRNA abundance was calculated as the Ct for amplification of agene-specific cDNA minus the average Ct for 18S expressed as a power of2 (2^(ΔCt)). Three individual gene-specific values thus calculated wereaveraged to obtain mean±SD. We choose ABCG2 as generic markers ofcorneal stromal stem cells, and keratocan, aldehyde dehydrogenase3A1(ALDH), prostaglandin D2 synthase (PTGDS), Keratan sulphate6-O-sulphotransferase (CHST6) and Pyruvate dehydrogenase kinase, isozyme4 (PDK4) as generic markers of keratocytes.

Immunostainning

The PEUU e-spun fibrous scaffolds seeded with hCSSCs were fixed in 4.0%paraformaldehyde in PBS at room temperature for 20 minutes, rinsed inPBS, and stored at 4° C. in PBS until further processing. Except the onefor keratocan, the fixed samples were incubated in 10 wt-%heat-inactivated goat serum (HIGS) at room temperature for one hour toblock nonspecific binding, rinsed in PBS, and incubated in 1-wt % bovineserum albumine (BSA)-PBS with mouse-monocloned primary antibodiesovernight at 4° C. in a sealed moist box. For immunostainning keratocan,the sample was firstly digested and blocked in 1-wt % bovine serumalbumine (BSA)-PBS with keratanase (0.5 unit/ml) for two hours at roomtemperature, rinsed in PBS, then stained by goat-monocloned anti-humankeratocan (a kind gift from Dr. Chia-Yang Liu) and incubated overnightat 4° C.

After three washes with PBS, secondary antibody Alexa Fluor488-conjugated goat anti-mouse or Alexa Fluor 543-conjugated donkeyanti-goat (1:2,500) (Invitrogen-Molecular Probes, Eugene, Oreg.,http://probes.invitrogen.com) together with4′,6-diamidino-2-phenylindole (DAPI) (0.5 lg/ml) (Roche MolecularBiochemicals, Indianapolis, Ind., http://www.roche.com) was added to thesamples, and incubated for 2 hours at room temperature. Omission of theprimary antibody served as a negative control. The stained wholemountswere placed in aqueous mounting medium (Thermo Fisher Scientific,Pittsburgh, Pa.) and examined using an Olympus FluoView FV1000 confocalmicroscope (Olympus, Tokyo).

Results

FIGS. 2A and 2D showed that surface topography of PEUU electro-spunsheet and cast film, respectively. The electrospun PEUU scaffoldfeatured highly oriented fibrils with nano-scale diameter (165±55 nm).In contrast, The PEUU cast film is comparatively flat and smooth.Although there are some local fluctuations induced by thermalperturbation during solvent evaporation, no particular orientation canbe noted. The cellular viability and morphology after cell seeding wasevaluated employing Calcein AM staining. As shown in FIG. 2B, thefluorescent viable hCSSCs were highly elongated and uniaxially alignedat the oriented fibrous PEUU scaffold. After three-day culture in stemcell growth medium (SCGM) with 2% bovine fetal serum, hCSSCs divided andproliferated to confluence. FIG. 2C shows that at latter time pointsproliferating cells maintain alignment. As expected, hCSSCs were notelongated on flat surface of PEUU cast film, and most of them showed thedendritic features (FIG. 2E. Similarly, cell morphology had littlechange with cell confluence after three-day culture in SCGM, as shown inFIG. 2F.

After three-day culture in SCGM, the scaffolds seeded with hCSSCs weretransferred into serum-free keratocyte differentiation medium (kDM).After six weeks incubation, analysis of the two types of cultures wascarried out. Firstly, we examined the influence of structured surface onthe gene expression of hCSSCs cultured in KDM. FIG. 3 showed the changesin gene expression by hCSSCs seeded on aligned nano-fibrous scaffold andcast film in KDM culture. In consistent with our previous observation(Du, Y., et al. Invest Ophthalmol Vis Sci 2007, 48, 5038-5045), hCSSCscultured in KDM down-regulated the expression of ABCG2, a typical markerpresent in many adult stem cells, and substantially upregulated severalgeneric markers of keratocytes, such as keratocan, aldehydedehydrogenase 3A1(ALDH), prostaglandin D2 synthase (PTGDS) and keratansulphate 6-O-sulphotransferase (CHST6). Specially, hCSSCs do not exhibitkeratocan, a typical gene marker expressed in corneal stromalkeratocyte. These observations revealed that hCSSCs were effectivelydifferentiated into keratocytes in KDM. The adult stem cell phenotype ofdifferentiated hCSSCs down-regulated much faster on aligned nano-fibrousscaffold than on cast film. Interestingly, keratocyte phenotypes ofdifferentiated hCSSCs on cast film have a little bit stronger expressionthan those on aligned nanofibrous scaffolds, however except keratocan.

Highly co-aligned molecules of Type-I collagen lead to strongbirefringence. In addition such aligned collagen features a second-ordernonlinear susceptibility because of its structural highnon-centrosymmetry, resulting in a strong second harmonic generation(SHG). Accordingly, the hCSSCs-secreted extracellular matrix (ECM) wasexamined by two-photon microscopy. As shown in FIGS. 4A-4B, although notstained, the second harmonic generation (SHG) signal (in red) is verystrong on both scaffolds when excited at λ_(ex)=840 nm. TheSHG-visualized ECMs secreted by hCSSCs are highly cohesive tissue-likemasses fibrous form. Specifically, the ECM on the aligned nano-fibrousscaffold was organized into fibril bundles that globally aligned in thewhole image from the top view. In contrast, the fibril bundles on castfilm are macroscopically random although regional alignment wasobserved.

With scanning electron microscopy, detailed surface morphologies ofhCSSCs and their deposited ECMs on the scaffolds from micron-scale tonano-scale were observed. On the aligned nano-fibrous PEUU scaffold, theseeded hCSSCs were elongated, and uniformly oriented in a preferreddirection, as shown in FIG. 5A. There are many dense fine fibers betweencells on the scaffold with alignment with high fidelity to hCSSCalignment. FIG. 4B demonstrates the detailed microstructures of thehCSSC-secreted fibril-like ECM. The longitudinal axes of the fibrilswere closely parallel to each other. Between the fibers, there arenumerous fiber-like side chains along the fibrils, which crosslink themtogether into an integrated collagen construct on the scaffold. As shownin the insert of FIG. 5B, a characteristic periodic banded structure wasconsistently observed similar to the D-band feature found in type-Icollagen fibrils.

In FIG. 5C a typical cell on the cast film substrate is seen with adendritic cellular morphology common to the film, but not the alignedfibrillar substrate. The secreted ECM on the cast film was also lessdensely packed. As shown in FIG. 5D, there is no preferred alignment ina macroscopic view. The distinct transverse banding pattern, thecharacter of Type-I collagen fiber, can also be seen along each fibrilas shown in the insert of FIG. 5D. Individual fibrils secreted fromhCSSCs on cast film did not appear to vary greatly from those on alignednano-fibrous sheets. Since SEM visualization only allowed assessment ofthe top layer of hCSSCs-secreted ECM organization, transmission electronmicroscopy was utilized to evaluate whether ECM distribution in thez-direction was consistent with physiological structures and variedbetween substrate types.

Transmission electron micrographs of the hCSSCs-secreted ECM incross-section are shown in FIGS. 6A-6H. Due to the defined structuralanisotropy, hCSSC-secreted ECM on aligned nano-fibrous scaffolds wasmicrotomed in orthogonally: along and across the fiber long axis. Forsamples cut across the fiber long axis, the hCSSC-secreted ECM wassandwiched by single cell layers and was ˜7-8 μm thick, as shown in FIG.6A. No cells were found in this gap. When viewed at higher magnification(FIG. 6B), all of the fibrils were found to be normal to the imagingplane. Within the ECM dense fiber clusters were present, with uniformfiber spacing, often as part of a “pseudo-hexagonal” lattice that ischaracteristic of native corneal stroma, (seen in the insert of FIG.6B). Fiber diameter (40.2±2.7 nm) and spacing (60.9±6.5 nm) within theclusters were analyzed by digital image processing with thedistributions summarized in FIGS. 6D and 6E. For samples cut along thefiber long axis, all fibers were found to be parallel to the viewingplane and each other and to be packed into clusters as shown in FIG. 6C.Digital analysis summarized in FIG. 6F revealed that the preferred angle(θ) was 89.8°±5.2°. As shown in the insert of FIG. 6C, uniform periodicbanding could clearly be seen along each fiber with a periodicity of 67nm, which is very close to the characteristic D-spacing of native Type-Icollagen. For hCSSCs cultured on cast film a sandwiched ECM was alsoobserved, as shown in FIG. 6G. However, in this case the fibers in the5˜6 μm thick ECM did not exhibit any preferred orientation. Under highermagnification (FIG. 6H), it can be seen that fiber size was not uniform;however the characteristic axial D-periodicity was observed along thesecreted fibers.

Also examined was the expression of collagens and proteoglycanstypifying the unique ECM of human corneal stromal tissue by whole mountimmunohistochemical staining. In FIGS. 7A and 7A′ it is seen that type-Icollagen was abundant in both secreted ECMs. Confirming electronmicroscopy analysis, the fibrous ECM deposited on the alignednano-fibrous sheet exhibited high alignment of type-I collagen. Inaddition, collagen V, collagen VI, keratan sulfate and keratocan werealso detected as shown in FIGS. 7B-7E. All of these representativecollagens and proteoglycans can also be found in hCSSC-secreted ECM onthe cast film, as shown in FIGS. 7A′-E′. However, on this film thefibrils demonstrated no preferred orientation. Structurally, the ECM onaligned nano-fibrous sheet better approximated the flattened lamellae ofcorneal stromal tissue, e.g. one single layer construct with highlyorderly uniform Type-I collagen fibrils. These results were inaccordance with the observations from two-photon microscopy.

Nano-structured collagen fiber is the fundamental building block ofconnective tissue and extracellular matrix (ECM). It is critical todesign and manipulate structured collagen-based ECM for the success intissue repair and regeneration medicine. In this study, we tentativelybioengineer 3-D orderly nano-structured collagen-fibril constructemploying tissue engineering strategy. Human cornea stroma is anavascular and acellular tissue, and an organization of corneal stromacollagen fibrils with mono-disperse fiber diameter and uniform localinterfibrillar spacing. Hierarchically, it consists of ˜200collagen-fibril lamellae, each about 1.5˜2.5 micrometer in thickness.The collagen fibers of each lamella are parallel with one another, butperpendicular to those of adjacent lamellæ produced by keratocytes. Thusorganizational and structural characteristics mean that corneal stromatissue is an ideal candidate for us to evaluate our strategy.

Cells, biomaterials-based scaffold, and biochemical and physio-chemicalfactors are the main factors for the success of tissue engineering. Innative corneal stroma tissue, keratocytes play the fundamental functionto sustain stroma tissue by secreting a spectrum of unique matrixproteoglycans. Unfortunately, they are not practical in tissueengineering because they will irreversibly lose their phenotypes anddifferentiate into fibroblast during their population expansion inserum-containing medium. The fibroblast has much less expression ofkeratocan and keratan sulfate, both of which are key molecules in theconnective tissue matrix of the cornea of the eye, and are believed toplay functional roles in collagen organization and the resulting cornealtransparency. Morphologically, pure fibroblast pellet culture was lackof abundant and organized collagen fibrils. In contrast, hCSSCs pelletculture showed the abundant extracellular matrix containing collagenfibrils. Specially, some aligned collagen fibrils can be observed at theperipherial region. However, most of them are in the form of amorphousspheroids. These observations demonstrated that hCSSCs cannotself-organize into 3-D orderly collagen-fibril construct by themselveswithout the guidance of an appropriate cellular environment.

Simply embedding cells in scaffolds usually result in poorly organizedECM (Langer, R., et al. Science 1993, 260, 920-926). Similarly,cell-secreted ECMs were in a form of an overall random orientation whencells were seeded on the substrate without features (Guillemette, M. D.,et al. Integrative Biology 2009, 1, 196-204). Although there are severalmethods to make cells orient in one preferred direction, micro-patternedsurface is the simplest one by providing the physical cue to cells.Gerecht et al. (Biomaterials 2007, 28, 4068-4077) revealed thatnano-structured PDMS surface coated by fibronectin can induce thereorganization of human embryonic stem cells' (hESCs′) cytoskeletoncomponents to modulate their morphology and proliferation. Guillemetteet al. (Integrative Biology 2009, 1, 196-204) found the multiple celllayers on the micro-patterned SEBS surface. Although the first celllayer aligned in the direction of the gratings, the second one variedwith cells. The second corneal fibroblast layer shifted angle of 53±8degree relative to the first layer, and the second smooth muscle celllayer features 39±4 degree shift. In contrast, the second layer ofdermal fibroblast had no orientation.

In this study, we fabricated aligned nano-fibrous scaffolds usingpoly(caprolactone)(PCL)-based poly(ester urethane) urea (PEUU) employingelectrospinning technique. PEUU shows high elasticity, biocompatibilitywithout toxic degradation products, and excellent processibility. It isan ideal scaffold material used for soft tissue engineering. The yieldedscaffold feature an aligned nano-structured surface, which effectivelyinduced hCSSCs to elongate and align following the PEUU fiberorientation as shown in FIGS. 2B and 2C. As a comparison, flat PEUU castfilm surface is incapable to guide cells' alignment, on which cellmorphology is dendritic.

The cell morphology and alignment directly affect the organization ofcell-secreted ECM. SEM micrographs showed that on the alignednano-fibrous PEUU sheet, hCSSCs-secreted collagen fibers were guided togrow along long axis of the elongated hCSSCs. As a comparison, thedendritic hCSSCs on the flat counterpart produced the ECM in anamorphous manner TEM further revealed the internal microstructures ofthese resulting ECM construct. On the aligned nano-fibrous PEUUscaffold, the secreted collagens self-organized in one preferreddirection in the whole construct, which was sandwiched by two cellmono-layers at the top and bottom. The fact that the surface observationis in accordance with the internal one means that collagen fibers growup in self-similar manner: the existent collagens guide the new one togrow up. Different from the constructs by corneal fibroblasts seeded onmicro-patterned SEBS substrate (Guillemette, M. D., et al. IntegrativeBiology 2009, 1, 196-204), the differentiated hCSSCs didn't make multicell-layer with an angle shift during the six-week culture. It may beassigned to our serum-free culture medium, resulting in much less celldivision. The controllable alignment of cells and cell-secret collagenfibrils is very significant of us to design and manipulate morecomplicated 3D-orderly collagen fibril construct employing bottom-upstrategy.

Another important observation is that collagen fibers in the constructfeature the uniform size and interfibrillar spacing. It is geneticallysimilar with the corneal stroma, although it is just one layerobservation. As a comparison, the collagen-fibril constructs made fromfibroblast lack of highly orderly features, though they show theapparent alignment on micro-patterned SEBS substrate (Guillemette, M.D., et al. Integrative Biology 2009, 1, 196-204) and Transwell™ PCmembrane (Guo, X. Q., et al. Invest. Ophthalmol. Vis. Sci. 2007, 48,4050-4060). Here, the phenotype of cells is a determinative factor. Asshown in FIGS. 7A-7E and 7A′-7E′, the collagen-fibril constructssecreted from hCSSC showed the strong positive expression of Type-Vcollagen, Type-IV collagen, keratocan, and keratin sulfate. Theydetermine collagen fibril size and interfibrillar spacing. Type-Vcollagen is believed to hybrid with Type-I collagen into the collagenfibers in human corneal stroma. The ratio of Type-I collagen to Type-Vcollagen determine the diameter of hybrid collagen fibrils. The normalratio in human corneal stroma is 4:1, resulting in 31±0.8 nm indiameter. The observed larger fiber diameter (40.2±2.7 nm) could be dueto the larger ratio of Type-I collagen to Type-V one. Type-VI collagenis another major collagen in human cornea besides its Type-I and Type-Vcounterparts. Keratocan is one member of the small leucine-richproteoglycan family, and the major keratan sulfate proteoglycans incorneal stroma. It is capable of binding collagen fibers, which resultsin its highly charged glycosaminoglycan (GAG) chains to extend out. Moreimportantly, its bi-functional characteristic renders it to crosslinkthe adjacent collagen fibers by protein moieties (This importantphenomenon was also observed in our SEM micrographs shown in FIGS.5A-5D). Thus makes it essential to regulate collagen fibril diameter,and particularly interfibrillar spacing. The absence of keratocan andother keratan sulfates usually leads to the disorganized fibril spacing.In our case, the interfibrillar spacing within the collagen cluster ispretty uniform (60.9±6.5 nm), although it is irregular out of thecollagen-fibril cluster. These typifying proteins can be found theircorresponding generic markers in gene expression profiles shown in FIGS.2A-2F. For instance, CHST6 codes for an enzyme necessary for theproduction of keratan sulfate. Its mutations lead to macular cornealdystrophy. KERA codes for the enzyme for the production of keratocan.Its mutations can cause cornea plana. These proteins, whose contents andstructures determine the collagen diameter and interfibrillar spacing,were generically species-dependent. Varying with specie, the collagendiameter can change from 24 nm (e.g. Fin whale) to 43 nm (e.g. Camel),and the interfibrillar spacing can change from 39 nm (e.g. Herring) to67 nm (e.g. Camel).⁶ Thus implies that collagen fibril diameter andinterfibrillar spacing can be tailored varying with the source ofcorneal stroma stem cells.

Besides cells and scaffolds, biochemical and physio-chemical factorsalso play a critical role in tissue engineering. Optimizing culturemedium is considered as an important way to effectively guide cells tosecrete and self-organize ECM in our preferred direction. In this case,serum-free culture medium effectively avoided losing the phenotypes ofkeratocytes. L-ascorbic acid-2-phosphate (A-2-P) is an important factorto enhance the synthesis and secretion of collagens by ketatocytes.Besides, It has been found that TGF-β_(n) (Transforming growth factorbeta) super-family in cornea, including TGF-β₁, TGF-β₂, and TGF-β₃, alsoplay an important role in modulating keratocyte proliferation,apoptosis, transcription and DNA condensation. Corneal TGF-β_(n)expression is significant for maintaining corneal integrity and cornealwound healing. For example, TGFβ₁ plays a vital role in scar formationin adult corneas. TGFβ₂ and TGFβ₃ are the important factors in cornealdevelopment and scarless wound healing during embryogenesis. Comparedwith the native corneal stroma tissue, our yielded construct is nottotally mature and perfect. TGF-β_(n) incorporation can be a potentialeffective way to improve 3D orderly collagen-fibril construct by hCSSCs,and accelerate the construct mature. The related research has beenconducting on the way.

The hCSSCs successfully secreted and self-organized the 3D orderlynano-structured construct comprising collagen fibrils on alignednano-fibrous sheet. The cells (residing at the top and bottom of ECM)can also be easily decellularized for us to finally obtain the cell-freecollagen construct. Conceivably, the structured collagen-based constructcan feature many great advantages, including supreme biocompatibility,structural anisotropy, structured surface with aligned collagen fibrils,well-matched modulus, etc. Employing bottom-up strategy, it is feasiblefor us to fabricate the complex structural organization extracellularmatrix (ECM) for tissue repair and regenerative medicine, e.g.bioequivalent of human corneal stroma.

In summary, we successfully prepared the orderly collagen nano-constructemploying tissue engineering strategy. We demonstrated that thescaffolds prepared from biodegradable PEUU provided an amenablemicroenvironment where human corneal stromal stem cells (hCSSCs) couldsecrete a type-I collagen-based ECM. Scaffold topography played thecritical role in initiating and guiding the organized expression of ahuman corneal stroma-like matrix by hCSSCs. Only the alignednano-fibrous scaffold resulted in the type-I collagen-based ECMcharacterized by one lamellae with oriented fibers as well as small anduniform fiber diameters and spacing. As a comparison, collagen fibrilsrandomly distribute on cast film with flat smooth surface.

More importantly, the detected expressions of collagen type-V, collagentype-VI, keratocan and keratan sulfate, which typify human cornealstromal tissue, indicated that the resulting ECM mimicked humanstroma-like tissue. These protein expression profiles also can be foundtheir corresponding generic markers employing RT-PCR technique.Genetically, fiber diameter and interfibrillar spacing can be tunedvarying with corneal stroma stem cells source. These striking resultsevidenced the feasibility that 3D orderly collagen-fibril nano-constructcan be generated by mimicking corneal stroma tissue employing tissueengineering strategy. These observations represent an important firststep of a bottom-up strategy to bioengineer complex collagen-basednano-biological construct for tissue repair and regenerative medicine.

Example 2

Follow-up studies were performed based on the results illustrated inExample 1. First, the electrospinning parameters were modified in orderto determine optimal rotational speeds in relation to the polymerconcentration in HFIP. Polymer preparation and electrospinning wasperformed essentially as described in Example 1. As shown in FIGS. 8 and9, fiber thickness and spacing was affected by electrospinningconditions, with all speeds and polymer concentrations yielding alignedfibers, but with a 5.0% PEUU in HFIP yielding optimal results.

FIGS. 10A and 10B depict a cell culture device used in the experimentsdescribed. Culture conditions were essentially as described in Example1.

The scaffold material prepared from the 5% PEUU in HFIP at 2000 rpm wasseeded with 8×10⁴ cells/cm² hCSSCs prepared as described in Example 1and cultured in stem cell medium for 3 days. KDM was added and the cellswere examined 3 and 6 weeks later. At three weeks after the addition ofthe KDM, the cells were confluent as determined by SEM. Cells weremicrotomed along their long axis and were seen by TEM to be elongatedand oriented (FIG. 11) and fiber structures characteristic of cornealstroma were seen when the cells were microtomed across their long axis(FIG. 12).

At six weeks after differentiation, the cells were examined bytwo-photon fluorescent microscopy showing highly-aligned cellstructures. As shown in FIG. 13, SEM micrographs show extensive cell andECM alignment. TEM shows uniform fiber size and characteristicpseudo-hexagonal lattice structures of corneal stroma (FIGS. 14A and14B) with fiber banding (FIGS. 14C and 14D). FIGS. 14E and 14F providegraphs showing fiber diameter and spacing that is very close to thatfound in human corneal stroma. FIG. 14G provides a graph showing fiberorientation.

As a control, a cast film was prepared using the same PEUU composition,and no cell or fiber alignment was seen (data not shown)Immunohistochemical analysis was performed on the case film cells, andcollagen, keratan sulfate and keratocan were present, but no alignedstructure was produced (data not shown). In a related experiment, 0.0%,5.0%, 10.0% and 20.0% type 1 collagen was added to the electrospun(performed as above) and cast substrates. Collagen in the templatescaffolding was observed to have a detrimental effect on cell alignmentand aligned scaffold formation over 12 weeks even though the initialtemplates appeared to have good quality aligned fiber structure (datanot shown).

In summary:

-   -   The aligned nano-fibrous scaffold prepared from biodegradable        PEUU provided an amenable microenvironment where hCSSCs could        secrete and organize a collagen type-I-based ECM into the        lamellae with oriented fibers.    -   The small and uniform fiber diameter and spacing were remarkably        similar to that found in the human corneal stroma.    -   The detected expression of collagen type-V, collagen type-VI,        keratocan and keratan sulfate indicated that the resulting ECM        mimicked human stroma-like tissue.    -   The incorporation of Collagen Type-I in scaffold has a negative        effect on the formation of oriented collagen type-I based        fibrous ECM.    -   The formed collagen type-I based fibrous ECM degenerated at the        long-term culture (i.e. >12 week), probably due to the        differentiated hCSSCs apoptosis (?).    -   These data represent an important first step of a bottom-up        strategy to bioengineer the human corneal stroma, and possibly a        complete bioequivalent human cornea.

Example 3—Adipose-Derived Stem Cells Differentiate to Keratocytes InVitro

Adipose-derived stem cells (ADSC) are an abundant population of adultstem cells with the potential to differentiate into several specializedtissue types, including neural and neural crest-derived cells. Thisstudy sought to determine if ADSC express keratocyte-specific phenotypicmarkers when cultured under conditions inducing differentiation ofcorneal stromal stem cells to keratocytes.

Methods

Cells and Materials:

Human corneal stromal stem cells (CSSC) and corneal fibroblasts (CF)were isolated and cultured as previously described (Du Y, et al.Secretion and organization of a cornea-like tissue in vitro by stemcells from human corneal stroma. Invest Ophthalmol Vis Sci 2007;48:5038-45 and Du Y, et al. Multipotent stem cells in human cornealstroma. Stem Cells 2005; 23:1266-75). Briefly, donor human corneas notusable for transplantation were incubated in 1.2 U/ml Dispase II (RocheDiagnostics, Pleasanton, Calif.) overnight at 4° C. Epithelial andendothelial cells were removed by dissection and debridement, and thestroma was minced into 2-mm cubes. Stromas were digested up to 3 h at37° C. in Dulbecco's modified Eagle's medium (DMEM) containing 1 mg/mlcollagenase type L (Sigma-Aldrich, St. Louis, Mo.). The resultingprimary keratocytes were cultured in a humidified atmosphere containing5% CO₂ in DMEM/F-12 (Sigma-Aldrich) with antibiotics for one week beforeharvesting for RNA. Stem cells from the stromal digest were expanded byculture at a density of 5×10³ cells/cm² in a stem cell growth medium(SCGM) consisting of low glucose DMEM:MCDB201 60:40 containing 2% fetalbovine serum (FBS), 10 ng/ml epidermal growth factor (EGF), 10 ng/mlplatelet derived growth factor BB (PDGF), 5 ug/ml insulin, 5 ug/mltransferrin, 5 ng/ml selenium, 200 U/ml LIF, andantibiotics/antimycotics. Fibroblastic differentiation was induced by 3or more passages in DMEM/F-12 with 10% FBS.

Human subcutaneous adipose tissue was obtained from patients undergoingelective lipoaspiration surgery with informed consent under a protocolapproved by the Institutional Review Board (IRB) of the University ofPittsburgh, consistent with the principles of the Declaration ofHelsinki. Adipose-derived stem cells were isolated by collagenasedigestions and differential centrifugation as previously described (AksuA E, et al. Role of gender and anatomical region on induction ofosteogenic differentiation of human adipose-derived stem cells. AnnPlast Surg 2008; 60:306-22). Primary adipose-derived cell mixtures werecultured at 5×10⁴ cells/cm² in SCGM. When the cells reached 80%confluency they were passaged 1:4 using trypsin. For flow cytometricanalysis trypsizined ADSC were incubated at 1×10⁶ cells/ml in DMEM with5 μg/ml Hoechst 33342 dye for side population cell sorting (Du Y, et al.Multipotent stem cells in human corneal stroma. Stem Cells 2005;23:1266-75)[23] and collected for further culture. Adipocytedifferentiation medium contained DMEM with 17 μM D-pantothenic acid(Sigma-Aldrich), 0.5 μM dexamethasone (Sigma-Aldrich), 0.2 nMtriiodothyronine (Sigma-Aldrich), and 1 μM ciglitazone (Enzo LifeSciences, Plymouth Meeting, Pa.). Chondrocyte differentiation wasinduced in DMEM/MCDB201, 2% FBS, 0.1 mM ascorbic acid-2-phosphate, 10-7M dexamethasone, 10 ng/ml recombinant transforming growth factor beta 1(TGFβ1; Sigma-Aldrich) and 100 μg/ml sodium pyruvate. Basal keratocytedifferentiation medium (KDM) contained Advanced DMEM (Invitrogen,Rockville, Md.) supplemented with 10 ng/ml fibroblast growth factor 2(FGF2) and 0.1 mM ascorbic acid-2-phosphate (A2P). Heparin-stripped,platelet-poor horse serum (HSHS) (Funderburgh J L, et al. ProteoglycanExpression during Transforming Growth Factor beta-inducedKeratocyte-Myofibroblast Transdifferentiation. J Biol Chem 2001;276:44173-8) was added as noted. Bovine corneal extract in DMEM/F-12 asan alternative to KDM was also used to compare keratocyte geneexpression after induction (Musselmann K, et al. Isolation of a putativekeratocyte activating factor from the corneal stroma. Exp Eye Res 2003;77:273-9). Antibodies used included anti-keratocan peptide antibody(KeraC)(Funderburgh J L, et al. Keratocyte phenotype mediatesproteoglycan structure: a role for fibroblasts in corneal fibrosis. JBiol Chem 2003; 278:45629-37) and J19 or J36 monoclonal antibodies tokeratan sulfate (Du Y, et al. Multipotent stem cells in human cornealstroma. Stem Cells 2005; 23:1266-75). Secondary antibodies for westernblotting, peroxidase-labeled antimouse and anti-rabbit IgG, were fromSanta Cruz Biotechnology (Santa Cruz, Calif.). For fluorescencestaining, Alexa Fluor 488 anti-mouse IgG and anti-rabbit IgG and nucleardye TO-PRO-3 were obtained from Invitrogen.

Side Population Cell Sorting:

ADSC were isolated as a side population on a high-speed cell sorter(MoFlo; DakoCytomation, Fort Collins, Colo.) using 350 nm excitation and450 nm emission in a method similar to that previously described forcorneal stromal stem cells (Du Y, Funderburgh M L, Mann M M, SundarRajN, Funderburgh J L. Multipotent stem cells in human corneal stroma. StemCells 2005; 23:1266-75). Verapamil was added to validate side populationisolation. After sorting, side population cells were cloned by limitingdilution, maintained in stem cell growth medium (SCGM) and passaged 1:3by trypsinization when subconfluent.

Pellet and Fibrin Gel Culture:

For pellet culture, 2×10⁵ passage-4 ADSC were collected in a conicalbottom 15-ml tube and centrifuged at 400×g for 5 min to form a pellet.The pellets were cultured in SCGM for 3 days, then transferred intovarious differentiation media which were changed every 3 days for up to3 weeks. For fibrin gel culture, 33 μl suspension of 12×10⁶ cells/mlpassage-4 ADSC were seeded into a fibrin gel consisting of 134 μl of 5mg/ml human fibrinogen (Sigma-Aldrich) and 33 μl of 100 U/ml bovinethrombin (Sigma-Aldrich). The gel formed in a cell culture incubator(37° C., 5% CO₂) for 1 h, and then SCGM containing 1 mg/mlε-amino-N-caproic acid (Sigma-Aldrich) was added for 3 days. The mediumwas replaced with KDM containing ε-amino-N-caproic acid at day 3 andchanged at 3-day intervals. Human corneal fibroblasts (CF) (Du Y, et al.Stem cell therapy restores transparency to defective murine corneas.Stem Cells 2009; 27:1635-42) were used as control for pellet culture andfibrin gel culture. The CF were cultured under the same conditions asADSC. Media were collected for western blot to detect the expression ofkeratocan and keratan sulfate after ion exchange isolation ofproteoglycans (described below). Cells from the same cultures were lysedto make RNA for RT-PCR or quantitative PCR or were fixed forimmunostaining.

Quantitative RT-PCR (qPCR):

Cell pellets and cells in fibrin gels were stored in a stabilizingreagent (RNAlater; Invitrogen, Austin, Tex.) for 1 day. RNA was thenisolated using the RNeasy mini kit (Qiagen, Valencia, Calif.), treatedwith DNase I (Invitrogen) and concentrated by alcohol precipitation.cDNA was transcribed from the RNA using SuperScript II reversetranscriptase (Invitrogen), following recommendations of themanufacturer. qPCR of cDNA was performed using assays containingfluorescent hybridization probes (TaqMan; Applied Biosystems, FosterCity, Calif.) or with direct dye binding (SYBR Green; AppliedBiosystems) as previously described (Du Y, et al. Secretion andorganization of a cornea-like tissue in vitro by stem cells from humancorneal stroma. Invest Ophthalmol Vis Sci 2007; 48:5038-45). Primers forSYBR assays were designed using online software (Primer 3) with thesequences shown in Table 2. Amplification of 18S rRNA was performed foreach cDNA (in triplicate) for normalization of RNA content. A negativecontrol lacking cDNA was also included in each assay. Relative mRNAabundance was calculated as the Ct for amplification of a gene-specificcDNA minus the average Ct for 18S expressed as a power of 2 (2^(−ΔΔCt)).Three individual gene-specific values thus calculated were averaged toobtain mean±SD.

TABLE 2 RT-PCR PRIMERS. GeneBank Gene Name Accession No.Primer sequence (53) Leptin NM_000230 Forward (SEQ ID NO: 1):TCCTGGATTCCTTTCCTTCA Reverse (SEQ ID NO: 2): CAATCGAGGAGGGCAGAATAKeratocan NM_007035 Forward (SEQ ID NO: 3): ATCTGCAGCACCTTCACCTTReverse (SEQ ID NO: 4): CATTGGAATTGGTGGTTTGA ALDH3A1 NM_001135168Forward (SEQ ID NO: 5): CATTGGCACCTGGAACTACC Reverse (SEQ ID NO: 6):GGCTTGAGGACCACTGAGTT 18S NR_003286 Forward (SEQ ID NO: 7): RibosomalCCCTGTAATTGGAATGAGTCCAC RNA Reverse (SEQ ID NO: 8): GCTGGAATTACCGCGGCT

Western Blotting:

Proteoglycans were recovered from culture media by ion exchangechromatography on microcolumns (SPEC-NH2; Agilent Technologies,Wilmington, Del.), as described previously (Du Y, et al. Multipotentstem cells in human corneal stroma. Stem Cells 2005; 23:1266-75).Proteoglycans were digested with a mixture of keratanase II andendo-β-galactosidase. Digested and undigested samples were run on a4%-20% SDS-PAGE gel, transferred to polyvinylidene difluoride (PVDF)membrane and subjected to immunoblotting with KeraC antibody againstkeratocan (Funderburgh J L, et al. Proteoglycan Expression duringTransforming Growth Factor beta-induced Keratocyte-MyofibroblastTransdifferentiation. J Biol Chem 2001; 276:44173-8) and antibody J36against keratan sulfate (Du Y, et al. Secretion and organization of acornea-like tissue in vitro by stem cells from human corneal stroma.Invest Ophthalmol Vis Sci 2007; 48:5038-45).

Histology:

Monolayer cells were rinsed briefly in phosphatebuffered saline (PBS),fixed for 12-15 min in 3% paraformaldehyde in PBS at room temperature,and rinsed in PBS. Oil red O (Sigma-Aldrich) was prepared at 0.5% inisopropanol, diluted to 0.3% in water and filtered before use. Cellswere stained with oil red O for 15 min and rinsed with 60% isopropanolfollowed by hematoxylin stain for nuclei. Bright-field micrography wasperformed with a 40× oil objective. Pellets and fibrin gels were rinsedbriefly in PBS, fixed for 15 min in 3% PFA in PBS at room temperature,rinsed in PBS, embedded in optimal cutting temperature embeddingcompound (Tissue-Tek OCT; Electron Microscopy Sciences, Hatfield, Pa.),frozen, and stored at −20° C. until they were cut as 8-μm sections on acryostat. Sections were hydrated in PBS before staining. Nonspecificbinding was blocked with 10% heat-inactivated goat serum. Sections wereincubated for 1 h at room temperature with primary antibodies. After tworinses in PBS, secondary antibodies and nuclear counterstain (TO-PRO-3;Invitrogen) were added for 1 h at room temperature. The samples werephotographed using a confocal microscope with a 20× oil objective(Bio-Rad Laboratories, Hercules, Calif.).

Results

Side population cells in ADSC: Human ADSC isolated by collagenase anddifferential centrifugation were labeled with Hoechst 33342 dye andanalyzed using flow cytometry to identify side population cells (FIGS.15A and 15B). This technique originally described by Goodell et al.(Isolation and functional properties of murine hematopoietic stem cellsthat are replicating in vivo. J Exp Med 1996; 183:1797-806) identifiescells that efflux the Hoechst 33342 dye as a result of the expression ofATP-binding cassette (ABC) transporter proteins. Side population cellsare present in small numbers in many tissues and have been found toexhibit adult stem cell-like properties (Zhou S, et al. The ABCtransporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells andis a molecular determinant of the side-population phenotype. Nat Med2001; 7:1028-34 and Challen G A, et al. A side order of stem cells: theSP phenotype. Stem Cells 2006; 24:3-12). FIG. 15A demonstrates that apopulation (defined by the box) amounting to less than 1% of the totalcells shows reduced Hoechst 33342 dye staining and a color shift towardblue in the cultured ADSC. When we preincubated with verapamil, aninhibitor of ABC transporter function, the population was eliminated(FIG. 15B). The presence of this characteristic side population suggeststhe presence of multipotent adult stem cells in the ADSC.

Differentiation potential of ADSC: To demonstrate multipotentdifferentiation potential, ADSC were expanded and grown in underconditions that induce differentiation to mature adipocytes in adultstem cells. FIG. 16A, FIG. 16B shows staining with oil red O, whichstains the characteristic neutral triglycerides and lipids inadipocytes. Well defined lipid droplets were present withindifferentiated cells. FIG. 16C, FIG. 16D shows the lack of oil red Ostaining in ADSC cultured in SCGM. Expression of the leptin geneencoding an adipokine secreted by mature adipocytes was increased inADSC after culture in ADM (FIG. 16C). Leptin was minimally expressed bykeratocytes, but was upregulated in multipotent stromal corneal stemcells as well as in ADSC.

ADSC were also cultured under conditions reported to induce chondrogenicdifferentiation. After 3 weeks in chondrocyte differentiation medium, asshown in FIG. 17A, ADSC secreted cartilage matrix as indicated bypositive toluidine blue staining for proteoglycans characteristic ofcartilage. In contrast, CF grown in pellet culture did not displaychondrogenic differentiation as evidenced by absence of toluidine bluestaining (FIG. 17B). Expression of keratocyte markers: Given the clearmultipotent nature of ADSC, we investigated the ability of these cellsto assume a keratocyte phenotype using methods previously successful indifferentiating human corneal stromal stem cells into keratocytes (Du Y,et al. Secretion and organization of a cornea-like tissue in vitro bystem cells from human corneal stroma. Invest Ophthalmol Vis Sci 2007;48:5038-45). ADSC were seeded in fibrin gels (FIG. 18A, FIG. 18B) andcultured as pellets (FIG. 18C, FIG. 18D) and transferred into KDM for 3weeks. Immmunostaining of ADSC cultures the showed presence of thestroma-specific ECM molecules keratocan and keratan sulfate in both thefibrin gels and pellets (FIGS. 18A-18E). ADSC in fibrin gels were moresparsely distributed (FIG. 18A, FIG. 18B) than in the pellet cultures(FIG. 18C, FIG. 18D). Expression of keratocan in ADSC incubated in KDMwas confirmed with RT-PCR using human keratocan primers (FIG. 18E). ADSCmaintained in SCGM alone did not express keratocan mRNA. In addition, weobserved (not shown) that ADSC in KDM formed extensive cell-cellcontacts similar to those connecting keratocytes. Cultures using bothfibrin gel and pellet methods induced ADSC differentiation intokeratocyte-like cells. After we established the differentiationpotential of ADSC into cells synthesizing keratocyte-specific proteins,we examined the effects of varying culture conditions on both keratocanand keratan sulfate expression levels. Using a combination of threedifferent culture media and fibrin gel or pellet culture, we found thatADSC in pellet cultures (FIG. 19C, FIG. 19D) had more consistentexpression of both keratocan and keratan sulfate at the protein and mRNAlevel than ADSC in fibrin gels (FIG. 19A, FIG. 19B). This was similar tohuman CSSC, which have elevated keratocan expression in pellet culturescompared to fibrin gel culture (FIG. 19A, FIG. 19B) (Du Y, et al.Secretion and organization of a cornea-like tissue in vitro by stemcells from human corneal stroma. Invest Ophthalmol Vis Sci 2007;48:5038-45). In fact, the level of keratocan mRNA in ADSC in pelletculture was similar to that of stem cells from corneal stroma (CSSC;FIG. 19D). Bovine corneal extract appeared to enhance differentiation ofthe ADSC but had little effect on CSSC (FIGS. 19A-19D, samples 6 and 9).

The gene ALDH3A1 codes for the widely distributed protein aldehydedehydrogenase (ALDH). In differentiated cells of the cornea, however,ALDH is exceptionally abundant, especially in keratocytes, where itmakes up as much as 40% of soluble protein (Jester J V, et al. Thecellular basis of corneal transparency: evidence for ‘cornealcrystallins’. J Cell Sci 1999; 112:613-22). Previously we observed ALDHto be markedly upregulated as CSSC differentiate to keratocytes (Du Y,et al. Secretion and organization of a cornea-like tissue in vitro bystem cells from human corneal stroma. Invest Ophthalmol Vis Sci 2007;48:5038-45) thus, we would expect ALDH upregulation if ADSC are adoptingkeratocyte phenotype. In FIG. 20 we documented strong upregulation ofthis corneal marker mRNA, particularly in the pellet cultures of ADSC.

Immunostaining of keratan sulfate (FIG. 21) and keratocan (FIG. 22)demonstrated that the mRNA increases documented in FIGS. 19A-19Dcorrelate with accumulation of these keratocyte-specific markers in theECM of the cultures. Consistent with mRNA levels, accumulation of thesematrix molecules was more evident in pellet cultures, and CSSC and ADSCgenerated similar amounts. This result was in contrast to CF which didnot consistently synthesize keratocan or keratan sulfate when grown ineither fibrin gels or pellets.

In this study we have shown that ADSC isolated from lipoasiprate havethe potential to differentiate in vitro into cells that synthesize andsecrete keratocyte-specific proteins as confirmed by bothimmunohistochemical and molecular evidence. Throughout our study we usedADSC grown and expanded at clonal density. These cells were cultured invarious differentiation conditions while maintaining their ability todifferentiate into adipocyte (FIGS. 16A-16E) and chondrocyte (FIGS. 17Aand 17B) lineages similar to results reported previously (Zuk P A, etal. Human adipose tissue is a source of multipotent stem cells. Mol BiolCell 2002; 13:4279-95). In addition, we show that ADSC grown inthree-dimensional fibrin gel and pellet culture systems supplementedwith appropriate differentiation medium can be induced to differentiateinto a keratocyte lineage (FIGS. 18A-18E, FIGS. 19A-19D, and FIG. 20).This differentiation is evidenced by the high levels of cornea-specifickeratocan mRNA and protein expression and the increased presence ofkeratan sulfate in the culture medium. Expression of aldehydedehydrogenase 3 family, member A1 (ALDH3A1), keratocan, and keratansulfate by ADSC was observed in several different media and in bothculture formats.

Our previous work showed that keratan sulfate is 10³ to 10⁶ fold moreenriched in cornea than any other tissue (Funderburgh J L, et al.Distribution of proteoglycans antigenically related to corneal keratansulfate proteoglycan. J Biol Chem 1987; 262:11634-40) and that synthesisof keratan sulfate by keratocytes is highly regulated in vitro and invivo (Long C J, et al. Fibroblast growth factor-2 promotes keratansulfate proteoglycan expression by keratocytes in vitro. J Biol Chem2000; 275:13918-23; Funderburgh J L, et al. Synthesis of corneal keratansulfate proteoglycans by bovine keratocytes in vitro. J Biol Chem 1996;271:31431-6; and Beales M P, et al. Proteoglycan synthesis by bovinekeratocytes and corneal fibroblasts: maintenance of the keratocytephenotype in culture. Invest Ophthalmol Vis Sci 1999; 40:1658-63).Keratan sulfate biosynthesis in vitro, therefore, represents the moststringent marker of the keratocyte phenotype yet described. Theobservation that ADSC can be induced to produce keratan sulfate is noveland presents the best evidence to date that these cells can adopt thekeratocytes phenotype. Keratocan is highly enriched in keratocytes, asis ALDH3A1. Neither of these proteins represents a unique corneal markerbut like keratan sulfate, both, are highly expressed in keratocytes andupregulated as CSSC differentiate to keratocytes (Du Y, et al. Secretionand organization of a cornea-like tissue in vitro by stem cells fromhuman corneal stroma. Invest Ophthalmol Vis Sci 2007; 48:5038-45).Upregulation of keratocan and ALDH3A1 mRNA simultaneously with synthesisof keratan sulfate by ASSC strengthens the argument that these cells areindeed differentiating to keratocytes.

High cell density, as occurs in pellet cultures, rather than dendriticcell morphology, appeared to positively influence keratocytedifferentiation potential. The pellet cultures produced a denser, moreabundant ECM with higher keratocan and keratan sulfate. This is similarto what we observed in human CSSC, which differentiate and expresshigher levels of keratocan and keratan sulfate in pellet cultures (Du Y,et al. Invest Ophthalmol Vis Sci 2007; 48:5038-45 and Funderburgh M L,et al. Keratocyte phenotype is enhanced in the absence of attachment tothe substratum. Mol Vis 2008; 14:308-17). Compared to fibrin gels, ADSCexpressed higher keratocan and keratan sulfate in pellet cultures (FIGS.19A-19D). Although pellet cultures clearly influenced keratocanexpression, the addition of extra supplementary factors such as horseserum and bovine extract did not appear to significantly enhance levelsof keratocan protein. Thus, keratocyte differentiation of ADSC appearsto be more dependent on the three-dimensional culture environment andless dependent on exogenous molecular supplementation. Growth ofdifferentiated keratocytes based on the architecture of the cultureconditions, rather than a complicated menu of biologically activemolecules, may be advantageous to the future isolation and production ofclinically useful cells while lowering the risk of contaminants fromadditions.

ADSC are an abundant and readily accessible source of multipotent adultstem cells with the desirable potential for autologous cell therapy,thereby presenting the potential for personal tissue engineering of thecorneal stroma. A recent study by Arnalich-Montiel et al.(Adipose-derived stem cells are a source for cell therapy of the cornealstroma. Stem Cells 2008; 26:570-9) demonstrated that humanlipoaspirate-derived cells could be transplanted into the corneal stromaof rabbits. Under these conditions, the ADSC did not elicit asignificant immune response, remained viable, and could be immunostainedfor ALDH and keratocan. The current study builds on these findings,using the more stringent keratocyte phenotypic marker keratan sulfateand defining in vitro conditions under which the keratocyte phenotype isexpressed by these cells. Understanding these conditions will allowdevelopment of use of ADSC in stromal bioengineering applications.

The immunomodulatory effects of ADSC are another important aspect oftheir potential use in cell based therapy. These effects have beenattributed to a lack of HLA-DR expression and active suppression of theproliferative T-cell response (McIntosh K, et al. The immunogenicity ofhuman adipose-derived cells: temporal changes in vitro. Stem Cells 2006;24:1246-53). ADSC have been shown to enhance dermal wound healing by thesecretion of a variety of soluble growth factors accelerating woundrepair and regeneration (Kim W S, et al. Wound healing effect ofadipose-derived stem cells: a critical role of secretory factors onhuman dermal fibroblasts. J Dermatol Sci 2007; 48:15-24). In addition,ADSC were shown to increase wound healing through differentiation intocell types capable of replacing and regenerating damaged tissue (AltmanA M, et al. IFATS collection: Human adipose-derived stem cells seeded ona silk fibroin-chitosan scaffold enhance wound repair in a murine softtissue injury model. Stem Cells 2009; 27:250-8). The biologic effectthese soluble factors have in corneal wounds remains to be determined,but could include production of the correct ECM and maintenance of akeratocyte phenotype. Particularly relevant to the environmentalexposures of the cornea, ADSC have been shown to provide protectiveantioxidant effects against chemically- and UVB-induced reactive oxygenspecies (Kim W S, et al. Evidence supporting antioxidant action ofadipose-derived stem cells: protection of human dermal fibroblasts fromoxidative stress. J Dermatol Sci 2008; 49:133-42 and Kim W S, et al.Antiwrinkle effect of adipose-derived stem cell: activation of dermalfibroblast by secretory factors. J Dermatol Sci 2009; 53:96-102).

Example 4—Adipose-Derived Stem Cells Cultured on Tissue Engineered 3-DOrderly Collagen Nanoconstruct Differentiate to Keratocytes In Vitro

Adipose-derived stem cells (ADSC) are an abundant population of adultstem cells readily isolated from human adipose tissue. ADSC havemultilineage potential and they are able to differentiate into fat,bone, cartilage, and muscle under lineage-specific culture condition.This study sought to determine if ADSC could be guided by alignednano-fibrous substance to biosynthesize the bioequivalent of humancornea stromal tissue employing corneal tissue engineering strategy. Weinvestigated if ADSC expressed keratocyte-specific phenotypic markerswhen cultured under condition inducing differentiation of cornealstromal stem cells to keratocytes.

Materials and Methods

Scaffold Preparation

Biodegradable Poly (ester urethane) urea (PEUU) was prepared. First,1,4-diisocyanatobutane and polycaprolactone-diol (PCL, M_(w)=2 kg/mol)were reacted in dimethyl sulfoxide (DSMO, Anhydrous Grade) for threehour at 75° C. with the aid of Tin 2-ethylhexanoate under the protectionof Ar₂ purge. After cooling down to room temperature, the oligomersolution was drop-wise added by 1,4-diaminobutane under vigorousstirring. After 18 hour reaction at room temperature, the polymersolution was precipitated in distilled water. Then the precipitant wassoaked in anhydrous 2-propanol for another 48 hours to remove DMSO andunbound monomers. The yielded polymer was incubated in anhydrous ethanolfor another 24 hours to remove water, and then further dried undervacuum at 40° C. for one week in order to remove water residual. Theyielded product is a white elastomer.

The oriented nanofibrous scaffolds were prepared by electrospinningBriefly, PEUU was dissolved in hexafluoroisopropanol (HFIP) undermechanical stirring at room temperature. The obtained polymer solutionwas fed by syringe pump (Harvard Apparatus) into a steel capillary(I.D.=0.047 inch) suspended on an aluminum wheel collector with 2-cm inwidth and 20-cm in thickness. A combination of two high-voltagegenerators (Gamma high Voltage Research) was employed with a highpositive voltage (+10 kV) to charge the steel capillary containingpolymer solution, and a high negative voltage (−5 kV) to charge thealuminum wheel collector with 20 cm in diameter. The distance betweenthe tip of the steel capillary and the top of the aluminum wheelcollector is 15 cm. The volume flow rate was set up as 1 ml/hr. The PEUUsolution was electrospun with 5.0 wt % concentration and rotationalspeed is 2000 rpm. The yielded fibrous scaffold is approximate 200micron thick.

Cell Cultures

The scaffold was punched into round discs with 25-mm in diameter to fitin 24-well culture plate. The discs were sterilized by UV exposure (254nm) in cell culture hood for 20 minute each side. Discs were fixed ineach well with plastic nuts and primary adipose-derived stem cells(ADSCs) were seeded on the scaffolds at a density of 1.6×10⁵ cells/well,which were incubated in stem cell growth medium (SCGM) containingDMEM/MCDB-201 with 2% fetal bovine serum (FBS), 10 ng/ml DMEM/MCDB-201with 2% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor, 10ng/ml platelet-derived growth factor (PDGF-BB), 5 μg/ml insulin, 5 μg/mltransferrin, 5 ng/ml selenous acid (ITS), 1,000 units per ml leukemiainhibitory factor (LIF), ×1 linoleic acid-bovine serum albumin (LA-BSA),0.1 mM ascorbic acid-2-phosphate, 10-8 M dexamethasone, 100 IU/mlpenicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin, and 1.25 μg/mlamphotericin B. Total of 48 scaffolds were prepared.

72 hours after incubation with SCGM, cells were exposed to keratocytedifferentiation medium (KDM, (KDM; advanced-MEM (Invitrogen) with 10ng/mL basic fibroblast growth factor (bFGF, Sigma-Aldrich), 0.1 mML-ascorbic acid-2-phosphate (A2P, Sigma-Aldrich), L-glutamine(1×GlutaMax™-1; Invitrogen), 50 μg/ml Gentamicin (Invitrogen), 100 μg/mlpenicillin (Mediatech, Inc.). KDM was changed twice a week for up to 6weeks. On day 14, 28, and 42, scaffolds were selected randomly from24-well plate and were subjected to two-photon fluorescent microscopy,scanning electron microscopy (SEM), transmission electron microscopy(TEM), western blotting, RT-PCR, and immunostaining.

Two-Photon Fluorescent Microscopy

Differentiated ADSC morphologies were observed with Two-photonFluorescent microscope. Scaffolds were randomly chosen from 24-wellplate on day 14, 28, and 42. Scaffolds were washed with PBS and werestained with CellTracker™ Green CMFDA (5-chloromethylfluoresceindiacetate) (Invitrogen) for 10 minutes. Samples were observed undertwo-photon fluorescent microscope.

Electron Microscopy

The morphologies of the differentiated ADSCs and their secretedextracellular matrix on the scaffold were investigated by ScanningElectron Microscope (SEM). The specimens were fixed in cold 2.5%glutaraldehyde (25% glutaraldehyde EM grade, Taab Chemical) in 0.1 M PBS(sodium chloride, potassium chloride, sodium phosphate dibasic,potassium phosphate monobasic, Fisher), pH=7.3. The specimens wererinsed in PBS, post-fixed in 1% Osmium Tetroxide (Osmium Tetroxidecrystals, Electron Microscopy Sciences) with 0.1% potassium ferricyanide(Potassium Ferricyanide, Fisher), dehydrated through a graded series ofethanol (30%˜90%-Reagent Alcohol, Fisher, and 100%-Ethanol 200 Proof,Pharmco), and hexamethyldisilazane (HMDS). The yielded sample wasinvestigated at 5 kV by Jeol JSM-6330F Scanning Electron Microscope(SEM) equipped with a digital camera.

The internal microstructures of the yielded ECM were investigatedemploying Transmission Electron Microscope (TEM). The sample was cutparallel and perpendicular to the alignment direction of PEUU fibrousscaffolds, respectively, in order to assess the influence of scaffoldsurface features on the ECM organization. The specimens were fixed incold 2.5% glutaraldehyde (25% glutaraldehyde EM grade, Taab Chemical) in0.1M PBS (sodium chloride, potassium chloride, sodium phosphate dibasic,potassium phosphate monobasic, Fisher), pH=7.3. The specimens wererinsed in 1×PBS, post-fixed in 1% Osmium Tetroxide (Osmium Tetroxidecrystals, Electron Microscopy Sciences) with 0.1% potassium ferricyanide(Potassium Ferricyanide, Fisher), dehydrated through a graded series ofethanol (30%-90%—Reagent Alcohol, Fisher, and 100%—Ethanol 200 Proof,Pharmco) and embedded in Epon (Dodecenyl Succinic Anhydride, NadicMethyl Anhydride, Scipoxy 812 Resin and Dimethylaminomethyl, Energy BeamSciences). Semi-thin (300 nm) sections were cut on a Reichart Ultracut,stained with 0.5% Toluidine Blue (Toluidine Blue O and Sodium Borate,Fisher) and examined under the light microscope. Ultrathin sections (65nm) were stained with 2% uranyl acetate (Uranyl Acetate dihydrate,Electron Microscopy Sciences, and methanol, fisher) and 1%phosphotungstic acid (Sigma-Aldrich), pH 3.2. The sections were examinedand photographed at 80 kV on Jeol 1011 transmission electron microscopeequipped with a digital camera.

Gene Expression

RNA of the differentiated ADSCs seeded on the PEUU scaffolds wasisolated using the RNeasy mini kit (Qiagen, Valencia, Calif.). RNA wastreated with DNAse I (Ambion) and was concentrated by alcoholprecipitation. RNA (200 ng) was transcribed to cDNA in a 50 μL reactioncontaining 1×PCR II buffer (Roche Applied Science, Indianapolis, Ind.),5 mM MgCl2, 200 μM dNTP mixture (Roche), 2.5 μM random hexamers(Invitrogen), 0.4 U RNase inhibitor, and 125 U SuperScript II reversetranscriptase (Invitrogen). Quantitative PCR of cDNA was performed usingassays containing fluorescent hybridization probes (TaqMan; AppliedBiosystems, Foster City, Calif.) or with direct dye binding (SYBR Green;Applied Biosystems) according to the manufacturer's instructions.Reactions were carried out on triplicate samples for 40 cycles of 15seconds at 95° C. and 1 minute at 60° C. after initial incubation at 95°C. for 10 minutes. Reaction volume was 20 μL. For TaqMan assays,reactions contained 1×Universal PCR Master Mix (Applied Biosystems), 1×gene mix, and 3.0 μL cDNA. For SYBR dye-based assays, the reactionscontained 1× PCR buffer (Applied Biosystems), 3 mM Mg′, 200 μM dATP,dCTP, dGTP, and 400 μM dUTP, 0.025 U/mL AmpliTaq Gold polymerase, 1.6 μLcDNA and forward and reverse primers at optimized concentrations.Amplification of 18S rRNA and GAPDH were carried out for each cDNA as aqualitative external control. A dissociation curve for each SYBR-basedreaction was generated on a real-time thermocycler (Gene-Amp ABI Prism7700 Sequence Detection System; Applied Biosystems) to confirm theabsence of nonspecific amplification. Amplification of 18S rRNA wasperformed for each cDNA (in triplicate) for normalization of RNAcontent. Relative mRNA abundance was calculated as the Ct foramplification of a gene-specific cDNA minus the average Ct for 18Sexpressed as a power of 2 (2^(ΔCt)). Three individual gene-specificvalues thus calculated were averaged to obtain mean±SD. Eight targetgenes including, ABCG2, aldehyde dehydrogenase 3A1(ALDH), AQP1,prostaglandin D2 synthase (PTGDS), Keratan sulfate 6-O-sulphotransferase(CHST6), keratocan, 18S, and GAPDH were chosen for PCR analysis.

Immunostaining of ADSC and Scaffold

Five PEUU e-spun fibrous scaffolds and ADSCs were randomly chosen from24-well plate and fixed in 4.0% paraformaldehyde in PBS at roomtemperature for 20 minutes. Samples were rinsed in PBS and stored at 4°C. in PBS for further treatment. Each fixed samples was incubated in 10wt-% heat-inactivated goat serum (HIGS) at room temperature for one hourto block nonspecific binding, rinsed in PBS, and incubated in 1-wt %bovine serum albumin (BSA)-PBS with mouse-monoclonal primary Collagen I,Collagen V, and Collagen VI antibodies overnight at 4° C. in a sealedmoist box. For detecting keratocan and keratan sulfate, the samples werefirstly digested and blocked in 1-wt % bovine serum albumin (BSA)-PBSwith keratanase (0.5 unit/ml) for two hours at room temperature, rinsedin PBS, then stained by goat-monoclonal anti-human keratocan (a kindgift from Dr. Chia-Yang Liu) or goat anti-human keratan sulfate,respectively, and incubated overnight at 4° C. After three washes withPBS, secondary antibody Alexa Fluor 488-conjugated goat anti-mouse orAlexa Fluor 555-conjugated goat anti-rabbit (1:2,500)(Invitrogen-Molecular Probes, Eugene, Oreg.,http://probes.invitrogen.com) together with4′,6-diamidino-2-phenylindole (DAPI) (0.5 μg/ml) (Roche MolecularBiochemicals, Indianapolis, Ind.) were added to the samples, andincubated for 2 hours at room temperature. Omission of the primaryantibody served as a negative control. The stained wholemounts wereplaced in aqueous mounting medium (Thermo Fisher Scientific, Pittsburgh,Pa.) and examined using an Olympus FluoView FV1000 confocal microscope(Olympus, Tokyo).

Immunoblot Analysis

KDM culture media were collected for 6 weeks. Proteoglycans wererecovered from culture media by ion exchange chromatography onmicrocolumns (SPEC-NH₂, Ansys Diagnositcs, Lake Forest, Calif.). Twosets of samples were run on 4%-20% SDS-PAGE gels, transferred topolyvinylidene difluoride (PVDF) membranes and each membrane wassubjected to immunoblotting with J19 antibody against keratan sulfate orwith KeraC antibody against keratocan.

Results

As shown above, ADSCs were differentiated into keratocytes when theywere seeded in fibrin gels. The expression of keratocan in ADSCs exposedto KDM was confirmed with RT-PCR using human keratocan primers (FIG.23A). As also shown (FIG. 23B), we confirmed that hCSSCs cultured on thenano-fibrous scaffolds upregulated several generic markers ofkeratocytes, including keratocan, aldehyde dehydrogenase 3A1(ALDH),prostaglandin D2 synthase (PTGDS) and keratan sulphate6-O-sulphotransferase (CHST6). In this example, we seeded ADSCs on thesame nano-fibrous scaffolds and incubated them with KDM for 6 weeks.After 6 weeks, gene expressions of ADSCs cultured in KDM were analyzedwith RT-PCR. ADSC cultured in KDM down-regulated the expression of ABCG2and AQP-1, typical gene markers expressed by many adult stem cells andsubstantially up-regulated expression of generic markers forkeratocytes, including keratocan, ALDH, PTGDS, and CHST6. GAPDH and 18Swere used as controls. Since ADSCs typically do not express keratocan,RT-PCR results suggested that ADSCs on the scaffolds were differentiatedinto keratocytes in KDM 6 weeks after incubation.

In order to confirm gene expressions of ADSCs on scaffolds, theretention of collagens and proteoglycans typifying unique ECM of humancorneal stromal tissue were examined by using whole-mountimmunostaining. Contrary to previous result, Type I, V, and VI collagenswere mostly intracellular and rarely formed ECM. FIG. 24 shows that TypeI collagen was mostly intracellular in the week 4 sample. There was norepresentative alignment of cells on the scaffolds, but rather cellswere spread out all over the scaffold without organization. In week 6sample, the number of cells increased dramatically and cells becamealigned in a particular orientation. Some positive stains for Type Icollagen were detected; however, it was not highly orderly uniformType-I collagen fibrils as shown in the hCSSC study. Similar trends wereobserved in Type V and VI immunostaining. In the week 4 samples, cellswere spread out and both Type V and VI collagens were mostlycell-associated. Some positive stains for Type VI collagen on ECM weredetected with week 6 sample; however, it was not highly ordered Type VIcollagen fibers. Additionally, the fibrils had no preferred orientation.Type V collagen was mostly detected within the cells and rarely on ECM.Keratan sulfate and keratocan were also cell-associated and were mostlydetected within the cells in both time points. In general, the number ofcells in all samples increased significantly over 6 weeks and cells werewell-aligned. Interestingly, there were two or three layers of cells ontop of the nano-fibrous scaffolds and each layer was formed with cellsoriented into one direction Immunostaining results were not completelyconsistent with RT-PCR data. Most of staining were observed within thecells and rarely seen on ECM. Although we detected some positivestaining for Type I, V, VI collagens on ECM, they were randomly spreadout between cells and were fragmented. In order to confirm our results,we decided to investigate ECM further with 2 photon microscopy.

The highly co-aligned molecules of Type-I collagen render the collagenfibers strongly birefringent. More importantly, they feature atremendous second-order nonlinear susceptibility because of itsstructural high non-centrosymmetry, resulting in a strong secondharmonic generation (SHG). Accordingly, the ADSC-secreted extracellularmatrix (ECM) was examined by two-photon microscopy. As shown in FIG. 25,although no staining, the SHG signal (in red) is very strong on bothscaffolds when excited at wavelength, λ_(ex)=840 nm. The SHG-visualizedECMs secreted by week 2 ADSCs were shown as fragmented fibers and someof them were intermingled together. Most of fiber-like structures werealigned in one orientation; however, there was no preferred orientationfor cell alignment. In the week 4 sample, the amount of SHG-visualizedECMs secreted by ADSCs increased and they formed tissue-like mass in theform of fibers, although they were short in length. Although fibrilswere aligned in one direction, cells were randomly spread out. In week6, the number of cells increased dramatically, and they were alignedvery well in one orientation. However, ECM secreted by cells was ratherunorganized and spread out on the scaffold only where no cells aligned.For week 2 and 4 samples, we used Sytox Green dye to stain only thenuclei of the cells. The nuclei were ellipsoidal and did not show apreferred alignment direction on both scaffolds. However, the nuclei inthe cells were not strictly related to the cell orientation. Therefore,next we used electron microscopy to investigate the relation betweencell orientation and ECM alignment.

Using scanning electron microscopy, we observed detailed surfacemorphologies of ADSCs and their deposited ECMs on the scaffolds frommicron-scale to nano-scale with increasing magnification. On the alignednano-fibrous PEUU scaffolds, the seeded ADSCs were elongated, anduniformly oriented into one preferred direction after 2 weeks ofincubation with KDM. There were many dense fibers between the cells onthe scaffolds. The majority of fibers were aligned in particularorientation, but some were branched out in many directions, intermingledwith each other and formed web-like structure. FIG. 26 revealed thedetailed microstructures of the ADSCs-secreted fibril-like ECM. Thefiber diameter was almost uniform, although the length of each fibercould hardly be accurately estimated. The longitudinal axes of thefibrils were closely parallel to each other. Between the fibers, therewere numerous fiber-like side chains along the fibrils, whichcrosslinked them. The number of cells on the scaffolds was increasedsignificantly over 6 weeks. Since SEM image represented only the toplayer of ADSCs-secreted ECM organization, it was hard to make aconclusion about collagen organization of ECM without looking at layersunderneath the top layer. Therefore, we needed to look at the internalmicrostructure with a transmission electron microscope.

Transmission electron micrographs of the ADSCs-secreted ECM incross-section are shown in FIG. 27. Due to its structural anisotropy,the ADSCs-secreted ECM on aligned nano-fibrous scaffold was microtomedin two orthogonal fashions: along and cross the fiber long axis. For thesample microtomed cross the fiber along axes, the ADSCs-secreted ECM wassandwiched by the single cell layers (data not shown). For the samplemicrotomed along the fiber long axes, all of fibers are parallel to theview plane as shown in FIG. 27. In week 2 sample, cells looked healthyand some fibrils that were aligned with cells in the same direction wereshown between cells. However, fibrils were rather short and looked likefragmented. When the sample was looked with higher magnification, fewobvious fibrils were shown with unique characteristic D-spacing of thenative Type-I collagen. In week 4 and 6 samples, number of cells andamount of fibrils increased, however, there was no particular pattern offibrils between cells. They were rather spread out all over the placeand formed clusters in random places. In week 6 sample, D-spacingbanding pattern appeared again in few fibrils which were aligned withcells; however, they were very short. Rest of fibrils were fragmented,clustered together, and looked like small dots. Based on the scale, thethickness of the cell layers on the scaffold was about 20-22 μm.

So far, only morphology or gene expression of ADSCs on the scaffolds hasbeen examined. Lastly, we wanted to investigate gene expression of theKDM media, in case any proteins were secreted from the cells into mediaduring 6 weeks. Western blotting of KDM collected media were done withJ19 antibody against keratan sulfate and with KeraC antibody againstkeratocan. Keratan sulfate expression was only detected from KDM mediaof first two weeks (FIG. 28). No keratocan expression was detected (datanot shown).

DISCUSSION

In this study, we have shown that ADSC, when they cultured onbioengineered 3-D orderly nano-structured collagen-fibril construct,have the potential to differentiate in vitro into cells that synthesizeand secrete keratocyte-specific proteins as confirmed by bothimmunohistochemical and molecular evidence. In order to fabricatealigned nano-fibrous scaffolds, we prepared scaffolding byelectrospinning poly (caprolactone)(PCL)-based poly(ester urethane) urea(PEUU). According to the previous studies, PEUU shows high elasticityand biocompatibility without toxic degradation products, and therefore,is ideal scaffolding material for soft tissue engineering. ADSCs wereseeded on the scaffold and were induced to elongate and align followingthe PEUU fiber orientation as they cultured with KDM for 6 weeks.

Given the clear multipotent nature of ADSC, we investigated the abilityof these cells to display keratocyte phenotypes when they were inducedwith KDM using methods previously successful in differentiating humancorneal stromal stem cells (hCSSC) into keratocytes. Previous studies inthe lab showed that keratan sulfate were much more enriched in corneathat any other tissue (Du, Y., et al. “Secretion and Organization of aCornea-like Tissue in Vitro by Stem Cells from Human Corneal Stroma.”Invest Ophthal Vis Sci 48 (2007): 5038-045). Therefore, keratan sulfatebiosynthesis in vitro, represents the most important marker of thekeratocyte phenotype. In addition to keratan sulfate, keratocan andALDH3A1 mRNA are both highly expressed in keratocytes and upregulated ashCSSCs differentiate to keratocytes. Therefore, upregulation ofkeratocan and ALDH3A1 with synthesis of keratan sulfate will providestrong evidence that ADSCs were differentiated into keratocytes.

We successfully showed that ADSC cultured in KDM down-regulated theexpression of ABCG2 and AQP-1, typical gene markers expressed by manyadult stem cells and substantially up-regulated expression of genericmarkers for keratocytes, including keratocan, ALDH, PTGDS, and CHST6.However, in further studies to examine the retention of collagens andproteoglycans typifying unique ECM of human corneal stromal tissue byusing the whole mount immunostaining, we observed that staining forkeratan sulfate and keratocan indicated that these proteins mostlyremained cell-associated. Although we observed positive staining of TypeI and VI collagens on the scaffold, they were unorganized and werealigned with neither nano-fibrils of the scaffold or the cells. SEM andTEM results also demonstrated that fibers between cells were short,unorganized, fragmented and were not fully structured to form ECM. Insome samples, we demonstrated that D-spacing banding pattern appeared infew fibrils which were aligned with cells; however, they were very shortand random to form ECM construct. Two-photon microscopy images showedthat cells were well aligned with scaffold fibrils, although ECMsecreted by cells were unorganized and appeared to be fragmented fibers.Two-photon microscopy was used to detect highly co-aligned molecules ofType I collagens. Collagens, which demonstrate structural highnon-centrosymmetry, will be resulted in a strong second harmonicgeneration. Lastly, we showed that there were no gene expressions ofkeratan sulfate or keratocan in KDM culture media we collected for 6weeks. Expression of keratan sulfate was detected during first twoweeks; however, gene expression decreased dramatically in 6 weeks.

Based on these results, we concluded that ADSCs cultured on PEUUnano-fibrous scaffold differentiated into keratocyte to some degree. Weconfirmed by RT-PCR that expression of genetic markers for keratocyteswere upregulated. We also confirmed positive staining for Type I and VIcollagens with immunostaining and presence of collagens on ECM with2-photon microscopy. Furthermore, SEM and TEM data showed that ADSCscultured with KDM generate some short, random, and fragmented fibersaligned or intermingled between cells and therefore, failed to form ECMconstruct. Although we showed increased gene expressions of genericmarkers of keratocyte, the true test of keratocyte function iselaboration of the highly organized, transparent ECM of the cornealstroma. Therefore, in order for ADSCs to be used in corneal cell therapyand tissue engineering, we need to further investigate methods to inducethese cells to generate ECM construct when they are differentiated intokeratocyte. One possible method will be changing chemical components ofKDM by adding another growth factor, such as transforming growth factorbeta.

In conclusion, our results provide novel evidence of potential of ADSCto adopt a keratocyte phenotype in vitro. Although more detailedmolecular characterization of the tissue elaborated by the ADSC will benecessary for clinical application, demonstration of non-ocular adultstem cells' ability to become corneal-like keratocytes in vitro withapplication of bioengineered nano-fibrous scaffolds opens an importantpotential for bioengineering of corneal tissue using autologous cells.

Having described this invention, it will be understood to those ofordinary skill in the art that the same can be performed within a wideand equivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any embodiment thereof.

We claim:
 1. A biological scaffold comprising: a plurality of layers ofaligned electrospun fibers of a biocompatible, biodegradable polymericcomposition, each layer having keratocytes seeded thereon and comprisingaligned extracellular matrix (ECM) deposited by the keratocytes, thepolymeric composition comprising: a polyurethane, a polyester, apolyether, a polyacrylamide, and/or a polycarbonate; a polyurethane, apolyester, a polyether, a polyacrylamide, and/or apolycarbonate-containing block copolymer, or a copolymer formed from oneor more acrylic monomers, acrylamide monomers, succinimide monomers,glycolide monomers, caprolactone monomers, dioxanone monomers, lactidemonomers, and/or carbonate monomers, and wherein the aligned fibers andaligned ECM of a first layer of the plurality of layers are arranged ata different angle with respect to the aligned fibers and aligned ECM ofa second layer of the plurality of layers that is adjacent to the firstlayer.
 2. The scaffold of claim 1, wherein the biocompatible,biodegradable polymeric composition comprises a poly(ester urethane)urea elastomer.
 3. The scaffold of claim 1, wherein the biocompatible,biodegradable polymeric composition comprises a polymer compositionhaving a Lower Critical Solution Temperature of 35° C. or less.
 4. Thescaffold of claim 3, wherein the biocompatible, biodegradable polymericcomposition comprises poly(N isopropyl acrylamide).
 5. The scaffold ofclaim 1, wherein the aligned fibers and aligned ECM of the first layerof the plurality of layers are arranged at a 20° to 90° angle withrespect to the aligned fibers and aligned ECM of the second layer of theplurality of the one or more layers that is adjacent to the first layer.6. A method of changing the refractive power of a cornea in an eye of apatient comprising: providing a template scaffold comprising: aplurality of layers of electrospun aligned fibers of a biocompatible,biodegradable polymeric composition, each layer having keratocyes seededthereon and comprising aligned extracellular matrix (ECM) deposited bythe keratocytes, the polymeric composition comprising: a polyurethane, apolyester, a polyether, a polyacrylamide, and/or a polycarbonate; apolyurethane, a polyester, a polyether, a polyacrylamide, and/or apolycarbonate-containing block copolymer, or a copolymer formed from oneor more acrylic monomers, acrylamide monomers, succinimide monomers,glycolide monomers, caprolactone monomers, dioxanone monomers, lactidemonomers, and/or carbonate monomers, and wherein the aligned fibers andaligned ECM of a first layer of the plurality of layers are arrangedorthogonally with respect to the aligned fibers and aligned ECM of asecond layer of the plurality of layers that is adjacent to the firstlayer allowing the aligned fibers to degrade, leaving a product ECMscaffold comprising the keratocytes and the ECM generated thereby; andonlaying or inlaying the product ECM scaffold in the cornea of a patientin need of an alteration of refractive corneal power.
 7. The method ofclaim 6 wherein the biocompatible, biodegradable polymeric compositioncomprises a poly(ester urethane) urea elastomer.
 8. The method of claim6 wherein the biocompatible, biodegradable polymeric compositioncomprises poly(N isopropyl acrylamide).
 9. The method of claim 6,wherein the aligned fibers and aligned ECM of the first layer of theplurality of layers are arranged at a 20° to 90° angle with respect tothe aligned fibers and aligned ECM of the second layer of the pluralityof the one or more layers that is adjacent to the first layer.
 10. Amethod of adding stromal tissue to a cornea of a patient, comprising:providing a template scaffold comprising: a plurality of layers ofelectrospun aligned fibers of a biocompatible, biodegradable polymericcomposition, each layer having keratocyes seeded thereon and comprisingaligned extracellular matrix (ECM) deposited by the keratocytes, thepolymeric composition comprising: a polyurethane, a polyester, apolyether, a polyacrylamide, and/or a polycarbonate; a polyurethane, apolyester, a polyether, a polyacrylamide, and/or apolycarbonate-containing block copolymer, or a copolymer formed from oneor more acrylic monomers, acrylamide monomers, succinimide monomers,glycolide monomers, caprolactone monomers, dioxanone monomers, lactidemonomers, and/or carbonate monomers, and wherein the aligned fibers andaligned ECM of a first layer of the plurality of layers are arrangedorthogonally with respect to the aligned fibers and aligned ECM of asecond layer of the plurality of layers that is adjacent to the firstlayer allowing the aligned fibers to degrade, leaving a product ECMscaffold comprising the keratocytes and the ECM generated thereby; andonlaying or inlaying the product ECM scaffold in the cornea of thepatient in need of corneal repair.
 11. The method of claim 10, whereinthe biocompatible, biodegradable polymeric composition comprises apoly(ester urethane) urea elastomer.
 12. The method of claim 10, whereinthe biocompatible, biodegradable polymeric composition comprises poly(Nisopropyl acrylamide).
 13. The method of claim 10, wherein the alignedfibers and aligned ECM of the first layer of the plurality of layers arearranged at a 20° to 90° angle with respect to the aligned fibers andaligned ECM of the second layer of the plurality of the one or morelayers that is adjacent to the first layer.
 14. The scaffold of claim 1,wherein the polymeric composition degrades in situ between one week andone year.
 15. The scaffold of claim 1, wherein the polymeric compositiondegrades within one month.
 16. The scaffold of claim 1, furthercomprising one or more keratocyte precursor cells.
 17. The scaffold ofclaim 1, wherein the aligned fibers have a diameter of 100-200 nm.